REVIEW ARTICLE Serine-arginine protein kinases: a small protein kinase family with a large cellular presence Thomas Giannakouros1, Eleni Nikolakaki1, Ilias Mylonis2 and Eleni Georgatsou2 Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Greece Laboratory of Biochemistry, Department of Medicine, School of Health Sciences, University of Thessaly, Larissa, Greece Keywords LBR; metabolic signalling; nuclear envelope; p53; PGC-1; protamine; spermatogenesis; splicing; SR protein; SRPK Correspondence E Georgatsou, Laboratory of Biochemistry, Department of Medicine, School of Health Sciences, University of Thessaly, Biopolis, 41110 Larissa, Greece Fax: +30 2410 685545 Tel: +30 2410 685581 E-mail: egeorgat@med.uth.gr (Received July 2010, accepted 26 October 2010) doi:10.1111/j.1742-4658.2010.07987.x Serine-arginine protein kinases (SPRKs) constitute a relatively novel subfamily of serine-threonine kinases that specifically phosphorylate serine residues residing in serine-arginine ⁄ arginine-serine dipeptide motifs Fifteen years of research subsequent to the purification and cloning of human SRPK1 as a SR splicing factor-phosphorylating protein have lead to the accumulation of information on the function and regulation of the different members of this family, as well as on the genomic organization of SRPK genes in several organisms Originally considered to be devoted to constitutive and alternative mRNA splicing, SRPKs are now known to expand their influence to additional steps of mRNA maturation, as well as to other cellular activities, such as chromatin reorganization in somatic and sperm cells, cell cycle and p53 regulation, and metabolic signalling Similarly, SRPKs were considered to be constitutively active kinases, although several modes of regulation of their function have been demonstrated, implying an elaborate cellular control of their activity Finally, SRPK gene sequence information from bioinformatics data reveals that SRPK gene homologs exist either in single or multiple copies in every single eukaryotic organism tested, emphasizing the importance of SRPK protein function for cellular life History of the discovery of the serine-arginine protein kinase (SPRK) family The first serine-arginine (SR) protein kinase to be purified and characterized was named SRPK1, for SR-protein-specific kinase [1,2] It was isolated during a search for the activity that phosphorylates SR splicing factors (also named SR proteins) during mitosis SRPK1 was shown to phosphorylate SR proteins in a cell-cycle regulated manner, to affect SR protein localization and to inhibit splicing when added in large quantities to a cell-free splicing assay [1,2] The SRPK1 cDNA was cloned, revealing that the Schizosaccharomyces pombe SRPK1 orthologue, Dsk1, had already been cloned and partially characterized as a kinase with cell cycle-dependent phosphorylation and subcellular localization [3] The SRPK1 and Dsk1 nucleotide sequencing identified a domain interrupting the kinase catalytic site into two structural entities, Abbreviations CDK, cyclin dependent kinase; Clk, CDK-like kinase; CK2, casein kinase 2; FOXO1, forkhead box protein O1; HBV, hepatitis B virus; HP1, heterochromatin protein 1; Hsp, heat shock protein; LBR, lamin B receptor; NRF-1, nuclear respiratory factor-1; PGC-1, peroxisome proliferator activated receptor c coactivator-1; RS, arginine-serine; SAFB, scaffold attachment factor B; SR, serine-arginine; SRPK, serine-arginine protein kinase 570 FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS T Giannakouros et al hence called ‘the spacer domain’, which is characteristic of the SR protein kinase family [3,4] Subsequently, Dsk1 was also shown to be a SR protein kinase phosphorylating and regulating the function of SR proteins [5–7] In 1998, the cloning of SRPK2 was reported almost simultaneously in mouse (together with mSRPK1) [8] and man [9] SRPK2 was found to be a SR-specific protein kinase highly homologous to SRPK1 It is structurally differentiated from SRPK1 by a prolinerich tract at its N-terminus and an acidic region in its spacer domain However, none of these elements had been related to a particular SRPK2-specific function until recently, when a study showed that sequences residing in the acidic domain of SRPK2 specifically interact with the pro-apoptotic arginine-serine (RS) domain-containing protein acinus [10] Moreover, the mRNA of SRPK2 was shown to have a different and more limited tissue distribution than SRPK1 mRNA [9] The cloning and characterization of the budding yeast Saccharomyces cerevisiae SR protein kinase Sky1 in 1999 came not only unexpectedly, but also as a challenge because the prevalence of uninterrupted genes and the lack of alternative splicing in this organism would not, at that time, account for a SR proteinspecific kinase [11] Sky1 was indeed shown to have the structural and functional characteristics of a SR protein kinase because it could not only phosphorylate mammalian SR splicing factors in vivo [12], but also native RS domain-containing S cerevisiae proteins, such as Npl3p, which is involved in mRNA export [13] This observation led to the discovery of the involvement of SRPKs in the regulation of additional steps of mRNA maturation and added to the current image of coupled transcript processing from the transcription to translation steps The cloning of SPK-1, the unique homolog of SRPK1 in Caenorhabditis elegans, revealed an essential function of the kinase in the germline development and embryogenesis of this organism [14] The underlying mechanism for this function has not yet been elucidated, although the finding that human SRPK1 is highly expressed in testis and phosphorylates protamine 1, a highly basic protein replacing histones during spermiogenesis, could be related with the observations in C elegans [15] SRPK1a, a product of the SRPK1 gene produced by alternative splicing, that retains an additional domain corresponding to an intron at its N-terminal region, was reported in 2001 [16] Interestingly, this domain is rich in proline residues reminiscent of the proline-rich SRPK2-specific track Additionally, the SRPK1a Serine-arginine protein kinases N-terminus was found to interact with the nuclear matrix protein scaffold attachment factor (SAFB) B1, and it was subsequently shown that SAFB proteins are inhibitors of SRPK1 and SRPK1a activity, functionally differentiating between the two kinases and further implicating SRPKs in subnuclear organization and chromatin regulation [17] Mouse SRPK3 was discovered in 2005, having been identified in a screen for target genes of the transcription factor myocyte enhancer factor [18] SRPK3 is expressed in a tissue-specific fashion in the heart and skeletal muscle and is required for normal muscle growth and homeostasis because Srpk3-null mice suffer from centronuclear myopathy [18] It has not been confirmed, however, whether SR kinase activity is required for these phenotypes and, if so, what substrates are affected The existence of the orthologue of mSRPK3 in humans has been postulated in an analysis of human chromosomal DNA methylation, although no studies are available for its expression or function However, the cDNA of the porcine SRPK3 has been cloned and shown to have a very limited and tissue specific expression in muscular tissue [19] Although Drosophila harbors several SRPK homologs, only two very recent studies refer to Srpk79D (as named by both groups), which is considered to be a product of the CG11489 gene in the Drosophila genome [20,21] It is interesting that Srpk79D displays tissue specific expression in neuronal tissue and is implicated in the development and growth of synaptic connections throughout the nervous system Finally, some SR protein kinases of lower organisms have also been cloned, adding to the picture of SRPK function and importance TcSRPK, the SR protein kinase of a protozoan, the parasite Trypanosoma cruzi, which displays trans- and cis-splicing and was cloned and characterized in 2003, functions as a bona fide SR protein kinase, indicating that the general control of eukaryotic mRNA processing evolved early during evolution [22] More recently, PSRPK, the SR protein kinase of Physarum polycephalum, a slime mold, has been cloned and characterized, especially with respect to its subcellular localization properties [23] Evolution of the SRPK gene family A simple search for genes (using the keyword ‘SRPK*’ at http://www.ncbi.nlm.nih.gov) returns approximately 90 hits for SRPK genes or putative SRPK genes in different eukaryotic organisms Some of these sequence entries have not yet been subjected to a final NCBI review and overlaps may exist between them that should be thoroughly examined In our preliminary FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS 571 Serine-arginine protein kinases T Giannakouros et al research, however, we have made some interesting observations that we note below to emphasize the significance of evolutionary-oriented studies on the sequences of the SRPK gene products The first intriguing observation is that the SRPK gene copy number of an organism does not appear to directly relate to its evolutionary scale For example, there exist fungi with one, two or even up to nine SRPK genes [S cerevisiae and S pombe with one gene (Sky1 and Dsk1, respectively); Candida albicans with two (QSAA48 and QS9Q27); Aspergilus niger with nine (A2QAE4, A2QB94, A2QC46, A5AB23, A2QWQ2, A2QX01, A2QX98, A2R2M0 and A2RSV1)]; plants with three genes (Ricinus communis; B9SRL4, B9S6V7 and B9SNS8); and insects with two (Culex quinquefasciatus; BOWGI3 and BOWRV4) or three genes [Drosophila melanogaster; CG8174, CG8565 and CG11489 (CG9085)], whereas mammals (rat, mouse, human, etc.) have three genes Additionally, as we have experienced from our own research and as mentioned in the two studies concerning Srpk79D in Drosophila melanogaster [20,21], there is no prominent one-to-one correspondence between the sequences of SRPK genes of evolutionary remote species The emerging image is reminiscent of independent SRPK gene duplications that have taken place at several time points during evolution in different species Accordingly, it is suggested that the SRPK genes are subjected to an evolutionary drive that demands multiple SRPK gene copies in almost each new emerging species One may observe evidence of the errors of the evolutionary ‘trial and error’ process operating through new SRPK genes: pseudogenes exist for both SRPK1 and SRPK2 in the human genome and also for SRPK1 in the mouse [24] Other loci identified by sequencing might also correspond to pseudogenes The second observation concerns the ‘spacer region’ of the SRPK proteins This sequence is SRPK familyspecific (as a serine ⁄ threonine kinase subfamily) and each family member harbors its own unique spacer It should be noted that the different ‘spacer regions’, in addition to being very different with respect to their primary sequence, are very diverse in length, and possibly function too, as indicated by the data presented further below Consequently, it is not unexpected that, for all the SRPK sequences we randomly examined from different kingdoms, the whole spacer sequence resides on a separate exon, suggesting that this domain may have evolved independently Another domain of interest that remains relatively unexamined from an evolutionary point of view is the N-terminal domain of the kinases It is highly specific between the SRPK family members, and few functions have been attributed to it It may be important to note 572 that it is this particular region of the mRNA that frequently swaps and alternates in the splicing phenomena that are beginning to be revealed in SRPK transcripts [16,20,21] Finally, it is interesting to note that SRPK2 contains a minor class of introns [25] Because the minor class of introns is often associated with many important genes that are evolutionarily conserved, it is likely that SRPK2 is evolving and regulated by a distinct mechanism from SRPK1 Function of the SRPKs As already noted, SRPKs phosphorylate their substrates at serine residues located in regions rich in arginine ⁄ serine dipeptides, known as RS domains The definition of a ‘typical’ RS domain is somewhat arbitrary and SRPKs have been shown to be able to phosphorylate scattered RS dipeptides if they conform to certain limitations [26–29] The specificity of these enzymes is remarkable because mutations of Ser to Thr or Arg to Lys in the RS domain completely abrogate phosphorylation [2,26] In the list of the RS domain-containing proteins, the SR proteins prevail, either as the originally identified ‘classical’ SR proteins invariably containing an RNA recognition motif or as ‘SR-like’ or ‘SR-related’ proteins also containing RNA binding domains (RNA recognition motif or other) Most of the SR splicing factors have been experimentally shown to be SRPK substrates in vitro and in vivo and it is to be expected that every SR protein could potentially be a SRPK substrate under particular cellular conditions Yet a recent study suggests that the human genome encodes for more than 100 RS domain-containing proteins [30], indicating that SRPKs may regulate diverse cellular functions through phosphorylation of many of these potential substrates Below, we review the SRPK impact on mRNA maturation and discuss the regulatory paradigms that have been characterized to a reasonable extent, including the replacement of histones by the arginine-rich protamines during spermiogenesis, the role of SPRKs in cell cycle progression and chromatin reorganization, and the function of SRPKs in the regulation of peroxisome proliferator activated receptor c coactivator (PGC)-1a in metabolic signaling SRPKs and mRNA maturation The involvement of SRPKs in the regulation of mRNA splicing was expected because SRPK1 was isolated as a SR splicing factor-phosphorylating kinase FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS T Giannakouros et al and the phosphorylation of SR proteins had been shown to be a prerequisite for spliceosome assembly and splicing in general [31–33] However, the exact contribution of the SRPKs in the different steps of mRNA maturation is not completely clarified (even up to this date) for several reasons First, the subcellular localization of SRPKs is cytoplasmic as well as nuclear, implying a more complex function for these kinases than the phosphorylation of only cytoplasmicor only nuclear-localized SR splicing factors SRPKs undoubtedly phosphorylate both, although under conditions that are strictly controlled Second, SR protein phosphorylation in the nucleus also takes place as a result of other families of kinases The cyclin dependent kinase (CDK)-like kinases (Clk) also phosphorylate RS domains but have a much broader specificity [26,34] Topoisomerase I has also been shown to phosphorylate SR proteins [35] but its role in SR protein function remains unexplored and, finally, and also relatively recently, Akt kinases have been shown to affect splicing by targeting RS domains [36] Third, the specific functions of the various SRPKs discovered in different organisms are just beginning to be addressed Serine-arginine protein kinases As already mentioned, concomitant with its purification, SRPK1 was shown to inhibit splicing in vitro when present in large quantities and to disassemble nuclear speckles when added in permeabilized cells [1] This and other in vitro experiments have implicated SRPKs in the phosphorylation of SR splicing factors and the regulation of splicing [2,9,37], although the first study to definitively attribute a role of a SRPK on SR protein function in vivo was carried out by Yeakley et al [12], which showed that when the unique SRPK of S cerevisiae (Sky1) is deleted, the interaction of SR proteins is prevented, and they are incapable of translocating into the nucleus Importantly, that study, which used mammalian splicing factors, showed for the first time that SRPK-mediated phosphorylation plays an important role in SR protein nuclear import and that not all SR splicing factors are affected identically Sequential studies with Sky1 and its SR-like protein substrate Nlp3p (which transports mRNAs out of the nucleus) have shown that Nlp3p needs to be phosphorylated to release the mRNA and be re-imported into the nucleus [13,38] In humans, shuttling splicing factors such as SF2 ⁄ ASF are phosphorylated in the cytoplasm by SRPK1 (Fig 1) and are subsequently Fig SRPK regulation and function in mRNA maturation During interphase, SRPKs are sequestered in the cytoplasm via their spacer domain and anchoring to various protein complexes, where they phosphorylate SR proteins and facilitate their nuclear import After stressinduced, cell cycle-dependent or other signalling, SRPKs translocate to the cell nucleus where, along with other SR protein kinases (Clks), they further modify their substrates found in nuclear speckles SRPK-mediated phosphorylation influences the dissociation of SR splicing factors from speckles, spliceosome assembly and splice site selection Dephosphorylation of SR splicing factors is required for splicing activity and their export to the cytoplasm In the nucleus, SRPKs can interact with nuclear matrix proteins such as SAFB Their export is the result of an as yet unidentified mechanism Black arrows indicate molecule reactions or movements Dashed lines indicate hypothetical molecule reactions or movements FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS 573 Serine-arginine protein kinases T Giannakouros et al transported to the nucleus by transportin-SR2, which specifically interacts with phosphorylated RS domains [39–41] In the nucleus, when not actively involved in transcript processing, SR proteins reside in nuclear speckles from which they are released when subjected to a new round of phosphorylation Although SRPKs are able to phosphorylate SR proteins residing in nuclear speckles, it is difficult to determine the exact roles of the different kinases involved in phosphorylation of SR splicing factors in the nucleus (Fig 1) The importance of the function of SRPKs for the splicing reaction in vivo first became apparent from the genetic approach of Dagher & Fu [42] in budding yeast, where it was shown that Sky1 interacts with proteins that affect 3¢ splice site selection Additionally, in co-transfection experiments, Nikolakaki et al [16] showed that both SRPK1 and SRPK1a are capable of altering the splicing of a tau minigene in a dose-dependent manner Moreover, it is interesting that, in humans, SRPK1 has been found to be associated with the U1-snRNP, which is involved in 5¢ splice selection [43], whereas SRPK2 was shown to be required for the formation of the U4 ⁄ U6-U5 tri-snRNP, which is involved in 3¢ splice site selection [44] Similarly, using siRNA, Hayes et al [45] have confirmed the role of SRPK1 in phosphorylating SR proteins in vivo and have connected the endogenous down-regulation of SRPK1 expression with alternative splicing of a particular transcript Finally, Zhong et al [46] clearly showed that when SR protein kinases enter the nucleus (in this case under a stress signal), phosphorylation of SR proteins is increased, verifying the nuclear action of SRPKs on SR splicing factors Accordingly, a study by Jiang et al [47] showed that when SRPK2 is phosphorylated by Akt in neuronal cells, it enters the nucleus and is able to phosphorylate the nonshuttling SR splicing factor SC35 As previously noted, SR proteins are implicated in a much broader spectrum of activities that accompany the life of an mRNA, in addition to the splicing process To function in mRNA export, SR proteins need to be underphosphorylated (Fig 1) On the other hand, SF2 ⁄ ASF does not have to leave the nucleus to exert its positive effect on mRNA nonsense mediated decay [48] An intact RS domain is required for this particular function, yet the impact of its phosphorylation state has not been addressed In addition, SF2 ⁄ ASF has been recently shown to be a translational activator of capped mRNAs in the cytoplasm [49]; however, no report on its state of phosphorylation was included in that study The participation of the SRPKs in these SR protein-dependent functions would be an interesting subject for future studies In 574 this respect, it should be noted that, in yeast, where the SR-like protein Npl3 was found to also affect translation (albeit by a different mechanism than SF2 ⁄ ASF in humans), this activity was shown to be Sky1-independent [50] The key role played by SRPKs in mRNA processing is particularly apparent in studies on pathological conditions, such as viral infection and tumor development The herpes simplex virus-1 protein ICP27 interacts with SRPK1, relocalizing it to the nucleus and affecting its function, resulting in lower total host splicing activity, and thus favoring the exit from the nucleus of the intronless viral mRNAs [51] The E1^E4 protein of human papilloma virus interacts with SRPK1 and can function as a substrate for the kinase The in vivo effects of this interaction are not known, however, nor is it known whether these putative effects would be exerted via the splicing machinery [52] A third virus found to be directly involved in SRPK function is hepatitis B virus (HBV) In HBV-infected cells, before the encapsidation of the virus genetic material, the unique viral core protein needs to be phosphorylated by a host kinase Although there are conflicting results as to whether the kinases responsible for this phosphorylation are SRPK1 and 2, there is agreement on the fact that the viral protein interacts with SRPK1 and that this interaction affects the HBV cell cycle [53,54] This as well as other evidence suggests that SRPKs may be potential pharmaceutical targets for the control of viral infection Hence, a small molecule, isonicotinamide compound, which is a relatively selective inhibitor of SRPK1 and (SPRIN340), was found to impair Sindbis virus propagation in cultured cells, although it is only variably effective on HIV-1 propagation [55] Interest in SRPKs as pharmaceutical targets also emerged from the observation that SRPKs show increased expression in tumors of pancreas, breast and colon [45,56], as well as in acute T-cell leukemia induced by human T-cell leukemia virus-1 [57] Accordingly, cell lines derived from pancreatic, breast and colonic tumors, when disrupted for the SRPK1 gene, display diminished cell proliferation, increased apoptotic potential and augmented sensitivity to the common chemotherapeutics gemcitabine and cisplatine Evidence has been provided that the results observed are effected through the splicing machinery [45] An inverse correlation has been documented, however, between the expression of SRPK1 and cisplatin sensitivity in yeast and in cells of germline origin, where down-regulation of SRPK1 confers resistance to cisplatin [58,59] These tissue-specific findings again point out the intricate and fine-tuned cellular networks regulated by SRPK activity FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS T Giannakouros et al SRPKs and spermiogenesis SRPK1, SRPK1a and SRPK2 are predominantly expressed in testis [9,15,16] Because of the numerous maturation stages that germ cells undergo, novel gene regulation strategies have been developed that provide for flexible gene expression and protein function and, among them, alternative splicing is particularly prevalent The overexpression of SRPKs in testis may therefore suggest a contributory role for these kinases in generating altered splice patterns across the developmental program of germ cells Yet the levels of SR proteins are not elevated in testis compared to other tissues [60,61], implying that the high levels of SRPKs are probably not just a result of changes in the splicing machinery The finding that P1 protamine from various organisms satisfy the substrate specificity requirements of SRPK1, coupled with the fact that most, if not all, of these proteins are known to be phosphoproteins [62,63], made them attractive candidate substrates of SRPK1 Consistent with this hypothesis, Ser10 and Ser8 (7RSQSRSR13) were identified as the in vivo phosphorylation sites of mono- and di-phosphorylated human P1 protamine [62] Indeed, SRPK1 was found to phosphorylate human P1 protamine efficiently [15] Protamines are highly basic, arginine-rich, low-molecular weight proteins that replace histones during the development of spermatids into spermatozoa, a process termed spermiogenesis [63] As a result of this exchange, the nucleosomal-type chromatin is transformed into a smooth fiber and compacted into a volume approximately 5% of that of a somatic cell nucleus [63,64] P1 protamine is the main member of the family and is conserved in all vertebrates, whereas P2 protamine has been described only in some species, including man, stallion, hamster and mouse [63] The deposition of protamines on sperm chromatin and the subsequent chromatin condensation are largely controlled by phosphorylation-dephosphorylation events Protamines are highly phosphorylated shortly after their synthesis and before binding to DNA [65] Phosphorylation of P2 protamine has been shown to be essential because deletion of the calmodulin-dependent protein kinase Camk4, which phosphorylates P2 protamine, impairs the deposition of P2 protamine on sperm chromatin, resulting in defective spermiogenesis and male sterility [66] Phosphorylation of P1 protamine by SRPK1 is required for the temporal association of P1 protamine with lamin B receptor (LBR), an inner nuclear membrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH2terminal domain [67] It is well known that RS Serine-arginine protein kinases domains mediate protein–protein interactions in a phosphorylation-dependent manner [68], assuming that only one of the two RS domains is phosphorylated Phosphorylation of the P1 protamine molecules in the cytoplasm on their way to the nucleus together with a lack of LBR phosphorylation is consistent with the observed predominant cytoplasmic localization of SRPK1 and the minimal RS kinase activity detected in the nucleus of germ cells [4,15] The association of P1 protamine with the nuclear envelope probably represents an important intermediate step before its deposition on sperm chromatin In this respect, Biggiogera et al [69] reported that protamines initially appear at the nuclear periphery, implying that the nuclear envelope might play a role in the replacement of transition proteins by protamines during spermiogenesis The detachment of P1 protamine from the nuclear envelope and its binding to DNA are probably achieved through its dephosphorylation (Fig 2) Consistent with this hypothesis, protamines were found mainly dephosphorylated in mature sperm chromatin [62,63] One possibility is that the nuclear envelope functions as a ‘working platform’ where additional modifications (e.g methylation) of P1 protamine take place These modifications may not only increase the affinity of P1 protamine for sperm DNA, but also may recruit specific molecules, such as heterochromatin protein (HP1), which were shown to be coupled to chromatin condensation and transcriptional silencing [64,70] A central question concerning P1 protamine is how its transportation into the nucleus is accomplished Conceivably, this may be mediated through an active transport mechanism, similar to histone H1 and transition protein 2, for which importin and importin 4, respectively, are known to be responsible for their translocation into the nucleus [71,72] Consistent with this hypothesis, it has been suggested that phosphorylation of the RS domain of the splicing factor ASF ⁄ SF2 by SRPK1 results in a conformational change that facilitates its interaction with the nuclear transport receptor transportin-SR2 (an importin-b family protein), thereby mediating the shuttling of this SR protein into the nucleus through the nuclear pore complex [41] In such a case, phosphorylation of P1 protamine by cytoplasmic SRPK1 may also promote its interaction with an as yet unknown importin family member, thereby facilitating its translocation into the nucleus The release of P1 protamine from importin may be mediated through its binding to LBR at the nuclear periphery Finally, SRPKs may have additional roles in spermatogenesis that need to be further characterized For FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS 575 Serine-arginine protein kinases T Giannakouros et al Fig A model illustrating the interactions between the NH2-terminal nucleoplasmic domain of LBR and P1 protamine At the beginning of spermiogenesis, the RS domain of LBR is unphosphorylated, allowing its association with phosphorylated protamine LBR may act as a docking site for the replacement of transition proteins (TP) by P1 protamine in certain chromatin layers that come close to the nuclear periphery Enzymes trapped in the inner nuclear membrane (INM) may also further modify the P1 protamine molecules, thereby facilitating their deposition on sperm chromatin The detachment of P1 protamine from the nuclear envelope and its tight binding to DNA is proposed to occur through its dephosphorylation, whereas, at the same time, a similar dephosphorylation event may trigger the dissociation of TP from sperm chromatin example, SRPK1 was reported to mediate the uptake of polyamines through an as yet unidentified signaling pathway [73] SRPKs, cell cycle progression and chromatin reorganization SRPKs have been characterized as cell cycle regulated kinases [1,3] This characterization was mainly based on the finding that SRPK1, as well as its fission yeast homolog, Dsk1, can translocate into the nucleus at the end of the G2 phase [3,4] In addition, SRPK1 activity, when assayed using SC35 or ASF ⁄ SF2 as substrate, has been reported to be approximately five-fold higher in extracts from metaphase compared to interphase cells [1] The break-up of the speckled pattern and the redistribution of splicing factors throughout the cytoplasm were initially considered as the main mitotic functions of SRPK1 [1] In this review, we discuss data associating SRPKs not only with additional mitotic events, but also with other functional aspects of the mammalian cell cycle Regulation of chromatin binding to the nuclear envelope Several macromolecular complexes are assembled by various integral proteins of the nuclear envelope that have been proposed to function as chromatin-anchorage platforms [74] LBR is one of the key factors that 576 has been implicated in chromatin anchorage and was shown to form oligomeric stuctures at the level of the nuclear envelope [75–77] The LBR–chromatin association is probably mediated by electrostatic interactions between the positively-charged residues of the N-terminal domain of LBR and the negatively-charged phosphate groups of DNA [78] The N-terminal domain of LBR harbors a RS domain, the phosphorylation of which not only reduces the positive charges, thereby weakening the interaction with DNA, but also may result in the disassembly of the oligomeric structure of LBR [79] The joint effect of the charge reduction and the conformational change may render the phosphorylated monomeric N-terminal domains unable to anchor the arrays of nucleosomes to the nuclear periphery It is well known that, during mitosis, the nuclear envelope breaks down and chromosomes dissociate from the inner nuclear membrane Already at prophase, binding of the membranous structures to chromosomes is weakened The RS domain of LBR is phosphorylated at the beginning of mitosis by nuclear-translocated SRPK1 and potentially by Akt and Clk kinases that may also target RS domains [26,36] Furthermore, the central mitotic kinase, cdk1, phosphorylates LBR at Ser71 [80], which is located just upstream of the RS repeats It is therefore possible that these combinatorial phosphorylation events may result in chromosome dissociation This idea is consistent with a previous study reporting that phosphorylation of LBR by mitotic extracts impairs chromatin association [81] FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS T Giannakouros et al Regulation of chromatin reorganization during G2 ⁄ M phase progression Another mode of action of SRPK1 related to its nuclear translocation at the G2 ⁄ M boundary involves chromatin reorganization A recent study demonstrated that two SR proteins, SRp20 and ASF ⁄ SF2, are released from mitotic chromosomes, during which the H3 tail is modified at Ser10 by the activated aurora kinase B, and reassociate with chromatin late in M phase [82] Hyperphosphorylation of these two SR proteins by SRPK1 was also found to significantly diminish their interaction with the H3 tail Intriguingly, dissociation of ASF ⁄ SF2 from phosphorylated histone H3 was required for the subsequent release of HP1 (a key constituent of interphase hetechromatin) from mitotic chromatin We propose that SRPKs (and potentially members of the Akt and Clk family of kinases) may have a key role at the beginning of mitosis by first mediating the detachment of peripheral heterochromatin from the inner nuclear membrane and, subsequently, the removal of HP1, thus leading to chromosome condensation (Fig 3) An even more intriguing possibility is that these phosphorylation events may also be applicable during interphase for fine-tuning gene expression It has been suggested by Misteli [83] that the differential regulation of gene expression might involve the inducible ‘potentiation’ of genomic loci, with subse- Serine-arginine protein kinases quent displacement from their chromosome territory and translocation to a transcriptionally silencing or activating microenvironment Because the coupling of chromatin domains to the nuclear envelope has been proposed to result in their transcriptional inactivation [84], and HP1 proteins are well-known constituents of ‘silent’ chromatin, the regulated nuclear translocation of SRPKs may contribute to the re-positioning and ‘unwinding’ of specific genomic loci, thus leading to their transcriptional activation Regulation of cyclin transcription SRPK2 has been implicated in the transcriptional regulation of two members of the cyclin family In hematopoietic cells, SRPK2 was reported to enhance cyclin A1 transcription [10], whereas, in neurons, it was shown to trigger cell cycle progression and induce apoptosis through regulation of cyclin D1 [47] Cyclin A1 is a member of mammalian A-type cyclins and is mainly expressed in male germ cells, being essential for the passage of spermatocytes into meiosis I [85] In addition to male germ cells, elevated levels of cyclin A1 expression have been detected in several leukemic cell lines as well as in hematopoietic stem cells and primitive precursors [86] Up-regulation of cyclin A1 by SRPK2 is accomplished through phosphorylation of the protein acinus that contains several RS domains and its subsequent redistribution from nuclear Fig Modulation of chromatin condensation at the beginning of mitosis by SR protein kinases The combined phosphorylation of the RS domain of LBR by nuclear translocated SRPK1 and the central mitotic kinase cdk1 (and potentially by Clk and Akt kinases) results in chromosome dissociation from the inner nuclear membrane A concomitant combined phosphorylation event [i.e phosphorylation of Ser10 of H3 by aurora B and phosphorylation of SR proteins ASF ⁄ SF2 and SRp20 (SR) by nuclear translocated SRPK1, and potentially by Clk and Akt kinases] results in HP1 release from mitotic chromatin, further facilitating chromatin condensation FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS 577 Serine-arginine protein kinases T Giannakouros et al speckles to the cytoplasm [10] Acinus was originally identified as a target of caspase-3, a cysteine protease involved in activating chromatin condensation and nuclear fragmentation during apoptosis; however, the role of this protein during normal cellular growth has not been determined [87,88] Acinus is phosphorylated by SRPK2 at Ser422, and acinus S422D, a SRPK2 phosphorylation mimetic, was shown to enhance cyclin A1 transcription, whereas acinus S422A, an unphosphorylatable mutant, was shown to block the stimulatory effect of SRPK2 Furthermore, siRNA-mediated down-regulation of acinus or SRPK2 resulted in cyclin A1 repression in leukemic cells and the cells were arrested at the G1 phase Interestingly, overexpressed FLAG-SRPK1 was unable to associate with and phosphorylate recombinant acinus, indicating that the interaction between acinus and SRPK2 is specific [10] To our knowledge, this is the first report of two SRPK family members exhibiting a differential recognition pattern towards an RS domain-containing protein Cyclin D1 functions as a mitogenic sensor and is one of the more frequently altered cell cycle regulators in cancers [89] It belongs to the family of mammalian D-type cyclins that are G1-specific Cyclin D1 associates with and allosterically activates CDK4 or CDK6, thereby promoting restriction point progression during the G1 phase [89] Terminally differentiated neurons are unable to reenter the cell cycle Aberrant cell cycle activation provokes neuronal cell death, whereas cell cycle inhibition increases neuronal survival SRPK2 triggers cell cycle progression in neurons and induces apoptosis through regulation of nuclear cyclin D1 [47] According to Jang et al [47], up-regulation of cyclin D1 in this system is not mediated through acinus phosphorylation but rather through inactivation of p53 More specifically, it has been proposed that SRPK2 phosphorylates and activates SC35 and, thus, it may inactivate p53 by blocking its phosphorylation at Ser15 [47,90] Interestingly, it has been also reported that SC35 affects transcriptional elongation in a genespecific manner [91] Thus, activation of SC35 may lead to down-regulation of specific genes, including p53 Because p53 represses cyclin D1 expression [92], down-regulation of p53 may also result in cyclin D1 up-regulation In this respect, it should be noted that SRPKs have been proposed to act as modifiers of the p53 pathway in Drosophila (Patent WO ⁄ 2002 ⁄ 099427: SRPKs as modifiers of the p53 pathway) More specifically, a genetic screen identified that a SRPK mutation enhanced cell death, as induced by the expression of p53 in the Drosophila wing Because Drosophila contains more than one Srpk gene, it remains unclear 578 whether the regulation of p53 activity is exerted by a specific SRPK (e.g the SRPK2 homolog) and, more importantly, whether this regulation is accomplished solely through SC35 SRPKs and metabolic signaling The PGC-1 family of coactivators mediates various environmental signals, thus regulating several metabolic pathways in a tissue-specific manner [93] Most importantly, the PGC-1 coactivators play a critical role in modulating glucose, lipid and energy homeostasis that become deregulated in metabolic diseases such as diabetes, obesity and cardiomyopathy The first member of the PGC-1 family, PGC-1a, was identified as a cofactor for peroxisome proliferator activated receptor c approximately one decade ago [94] PGC-1a activity is modulated by a large number of post-translational modifications, including phosphorylation by several kinases, acetylation and deacetylation by GCN5 and silent information regulator 1, respectively, as well as O-GlcNAcylation by O-GlcNAc transferase [95] PGC-1a contains a RS domain that links insulin signal transduction to the repression of gluconeogenesis [96] This link is mediated through phosphorylation of the RS domain that renders PGC-1a unable to coactivate the forkhead transcription factor forkhead box protein O1 (FOXO1), which is the main nuclear receptor controlling the glyconeogenic program [96–98] To date, two kinases have been implicated in the phosphorylation of the RS domain: Akt2 and Clk2 [97,98] Akt2 phosphorylates only Ser570, which is the last serine in the first repeat of RS dipeptides (RSRSRSFSR) [97], whereas Clk2 probably phosphorylates the entire RS domain [98] A central question that arises is whether PGC-1a is also phosphorylated by members of the SRPK family, and whether this phosphorylation can repress its transcriptional activity It was previously shown that SRPK1 can phosphorylate in vitro the RS domain of PGC-1a [79], although a similar phosphorylation event has not yet been shown to occur in vivo We anticipate that this type of phosphorylation may also take place in vivo and not only by SRPK1, but also by other members of the SRPK family, to an extent proportional to the expression levels of SRPKs in liver Another important issue is the response to insulin Akt2 is an insulin-responsive kinase, whereas it was shown to phosphorylate Clk2 at Thr343, leading to an increase of Clk2 protein stability and therefore activity [98] Clk2 was therefore suggested to function as an insulin-induced gluconeogenic repressor Yet Akt FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS T Giannakouros et al kinases can also phosphorylate SRPK2 at Thr492 and mediate its nuclear translocation [47], thus making SRPK2 an insulin-responsive kinase as well It is still unknown whether insulin has any effect on SRPK1 and ⁄ or on SRPK1a, either directly through phosphorylation by Akt kinases or through another indirect signalling mechanism In this respect, it should be noted that SRPK1a contains two LXXLL motifs [16] that are assumed to facilitate the interaction of different proteins with nuclear receptors All these phosphorylation events may act in a complementary fashion (Fig 4A), thus constituting a fine-tuning mechanism Serine-arginine protein kinases that modulates the interaction of PGC-1a with various transcription factors and allows the expression of specific gene sets in different physiological settings PGC-1a also stimulates mitochondrial biogenesis through coactivation of nuclear respiratory factor-1 (NRF-1) [99] Indeed, PGC-1a expression in both muscle and fat cells activates the expression of several genes of the oxidative phosphorylation pathway, including cytochrome c oxidase subunits II and IV, and ATP synthase Although the RS domain of PGC1a mediates its interaction with FOXO1, the 200-400 amino acid region of PGC-1a is responsible for the Fig Regulation of PGC-1a transcriptional activity by its RS region (A) Akt2, Clk2 and potentially SRPK2 phosphorylate various serine residues within the RS region of PGC-1a, thus impairing its interaction with FOXO-1 to a different extent each time Akt2 phosphorylates only Ser570 (purple), SRPK2 (and possibly other SRPKs as well) may phosphorylate the serines within the three repeats of RS dipeptides (blue), whereas Clk2 probably phosphorylates the entire RS domain (red) Akt2 is activated by insulin, whereas it phosphorylates Clk2 at Thr343, leading to an increase of Clk2 protein stability and activity Akt kinases also phosphorylate SRPK2 at Thr492 and mediate its nuclear translocation, thus rendering SRPK2 molecules available to phosphorylate nuclear PGC-1a It is not clear yet whether there is further cross-regulation between SRPK, Clk and Akt kinases The stoichiometry of PGC-1a phosphorylation (i.e the number of protein molecules per cell that are phosphorylated) and also the exact serines of the RS region that are modified in each molecule may constitute a fine-tuning mechanism that regulates the transcription of gluconeogenic genes and mediates PGC-1a responsiveness to insulin (B) A more permanent inactivation of the RS domain (in muscle and fat cells) may be achieved through binding of p32 protein A central function of p32 protein is to associate with and impair the phosphorylation of RS domains p32 protein may obstruct the interaction of PGC-1a with FOXO-1 that requires the RS domain, thus allowing the available PGC-1a molecules to interact with NRF-1 and promote the transcription of specific genes involved in oxidative phosphorylation FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS 579 Serine-arginine protein kinases T Giannakouros et al PGC-1a ⁄ NRF-1 interaction Thus, the ability of PGC1a to interact with different transcription factors allows for the coordinated expression of gene sets in specific cellular contexts and in response to specific signals A recent study adds another piece to the puzzle and further strengthens the hypothesis that SRPKs may be actively involved in the phosphorylation of PGC-1a Fogal et al [100], extending previous observations [101], reported that p32 protein plays a decisive role in maintaining mitochondrial oxidative phosphorylation Knocking down p32 expression in human cancer cells resulted in a reduced expression of oxidative phosphorylation-related polypeptides and shifted the cell metabolism from oxidative phosphorylation to glycolysis p32 is an ‘all-around’ cellular protein found in the nucleus, cytoplasm, mitochondria and cell surface with essentially unknown physiological role(s) [102] Yet p32 was reported to bind the RS domains of both ASF ⁄ SF2 and LBR and inhibit the phosphorylation of these molecules by SRPKs [67,103,104] Even though it remains to be proven, we speculate that p32 protein drives PGC-1a activity toward specific gene sets involved in oxidative phosphorylation by obstructing its interaction with FOXO1, thus allowing the available molecules to interact with NRF-1 (Fig 4B) Regulation of the SRPK family members SRPKs have been considered to be constitutively active kinases because the expression of SRPK family members in bacteria, which lack the post-translational modification machinery of eukaryotic cells, has shown that they are able to efficiently phosphorylate their substrates [26,29,105] Furthermore, co-expression of SRPK1 and its substrate SF2 ⁄ ASF in Escherichia coli results in the phosphorylation and splicing activity of the latter [106] In support of these findings, structural and biochemical studies on SRPK1 and Sky1p have shown that their kinase core domains can adopt, through a network of connections, an active conformation even when they are extensively mutated or their N-terminal and spacer domains are truncated, providing more evidence of their constant activity and substrate phosphorylation mechanisms [107,108] It might be expected that this family of kinases is somehow regulated Reports from various studies agree that the determining factor in SRPK regulation is their subcellular partitioning It has been demonstrated that SRPKs are primarily located in the cytoplasm of interphase cells and, to a lesser extent, in the nucleus [4,9,16,37] This cytoplasmic sequestration mainly depends on the existence of the unique and 580 divergent spacer sequence in each family member that divides the conserved catalytic kinase domains into two halves As initially reported for Dsk1 and Sky1p in S pombe and S cerevisiae, respectively, deletion of the spacer domain results in their nuclear translocation, with no apparent loss of activity Studies have shown that this sequence acts like a cytoplasmic anchor critical for SRPK regulation because the constant accumulation of mutant Sky1p in the nucleus provokes inhibition of cell growth [3,11] In addition, removal of the spacer domain in SRPK1 ⁄ forces their displacement to the nucleus, causing the aggregation of splicing factors and possibly affecting gene expression [4] The cytoplasmic anchoring of SRPKs has recently been shown to be mediated by their association with specific members of molecular chaperones (Fig 1) Thus, direct interaction of SRPK1 with cochaperones Aha1 and heat shock protein Hsp40 mediates the formation of a complex with the Hsp70 ⁄ Hsp90 machinery [46] Furthermore, SRPK2 directly associates with the -b and -e isoforms of 14-3-3 family of proteins in an Akt phosphorylation-depended manner in the cytoplasm of neuronal cells [47] The interaction of SRPK1 with the molecular chaperones could be modulated by signal(s) resulting in the release and subsequent translocation of the kinase to the nucleus One option is that SRPKs may be posttranslationally modified in response to signaling In this respect, a previous study indicated that SRPK1 is phosphorylated and partially activated by casein kinase (CK2) [109] However, it remains to be determined whether CK2 has any effect on the nuclear translocation of the kinase Furthermore, Akt was proposed to mediate the nuclear translocation of SRPK2 by phosphorylating it at Thr492, whereas 14-3-3 molecules were shown to interact with Akt-phosphorylated SRPK2 and inhibit its nuclear translocation [47] Of note, the major CK2 phosphorylation site (SDDD, Ser51 in human SRPK1) is conserved among SRPK family members, whereas the Akt site (HDRSRTVS, Thr492 in human SRPK2) is not, and probably represents a SRPK2-specific mode of regulation A second option is that the nuclear translocation of the kinases may be accomplished through the reorganization and ⁄ or modification of specific components of the chaperone complex Supporting this option is the demonstration that inhibition of the ATPase activity of Hsp90 with 17-AAG results in partial translocation of SRPK1 in the nucleus [46] Finally, as already noted, the ICP27 protein of HSV-1 interacts with SRPK1 and promotes its translocation to the nucleus [51] Yet none of the above signals is cell cycle-regulated; therefore, the signal triggering the nuclear translocation of FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS T Giannakouros et al SRPK1 at the G2 ⁄ M boundary remains to be determined The molecular mechanism that mediates the nuclear export of SRPKs also remains obscure Only the fission yeast Dsk1 has been shown to harbor an active nuclear export signal inside its spacer region that is recognized by CRM1 because Dsk1 export is impaired by leptomycin B treatment [110] Functional nuclear export signal sequences that may explain efficient transport through the nuclear membrane remain to be identified in other SRPK family members In addition to subcellular partitioning, other modes of SRPK regulation also exist Despite the fact that unmodified kinase molecules are active, it was shown that CK2-mediated phosphorylation of SRPK1 enhances its activity by six-fold [109] The primary CK2 target site (Ser51 at SRPK1) was also found to be modified in vivo by MS in kinome-wide phosphoproteomics studies in HeLa cells [111–113] These reports also indicate the existence of additional phosphorylation sites on SRPK molecules (http:// www.phosphosite.org/homeAction.do; keywords ‘SRPK1’ and ‘SRPK2’), suggesting that there is more than one unidentified signal, which could affect either their activity or shuttling through the modulation of their interaction with other proteins Another aspect of the modulation SRPK activity involves transient interaction with nonshuttling protein complexes It was recently shown that both SRPK1 and SRPK1a could directly interact with SAFB1 and SAFB2, albeit with different affinities This association does not depend on the spacer domain, as was shown for other protein complexes of SRPK1, but rather on the N-terminal and core kinase domains Interaction with SAFB molecules impaired the catalytic activity of SRPK1 ⁄ 1a, whereas the nuclear subfraction of the kinases, which was found to be associated with the nuclear matrix via SAFB proteins, was inactive [17] Given that SAFB proteins are also sequestrated in stress-induced subnuclear bodies, along with splicing factors and RNA molecules in response to stress [114,115], it is intriguing to consider that their interaction with SRPK1 ⁄ 1a and subsequent inactivation of the kinases could provide an additional mechanism of controlling SRPK activity when the cell needs to react promptly to a variety of signals Conclusions In conclusion, the accumulated knowledge on SRPK function not only enlightens many aspects of their influence on fundamental cellular mechanisms, but also raises questions that need to be addressed in future Serine-arginine protein kinases studies to obtain insight into their role in the cell These include: l The conformational changes induced by the phosphorylation of the RS domain l The exact share the SRPKs hold amongst the other kinases that also recognize RS dipeptides, such as the Clk and Akt family of kinases, as well as the cross-regulation between them l The specific role(s) of each one of the SRPK family members and the significance of the ‘spacer domain’ for the functional properties and the particular regulation of each kinase l The extra- and intracellular signals that regulate SRPK function Further investigations into the above issues, in light of a thorough examination of the evolutionary history of the SRPK genes, will help to unveil the functional presence of the SRPK family in cellular life Acknowledgements We thank John Georgatsos for critically reading the manuscript This work was supported by grants from the Greek General Secretariat of Research and Technology and the Greek Ministry of Education References Gui JF, Lane WS & Fu XD (1994) A serine kinase regulates intracellular localization of splicing factors in the cell cycle Nature 369, 678–682 Gui JF, Tronchere H, Chandler SD & Fu XD (1994) Purification and characterization of a kinase specific for the serine- and arginine-rich pre-mRNA splicing factors Proc Natl Acad Sci USA 91, 10824–10828 Takeuchi M & Yanagida M (1993) A mitotic role for a novel fission yeast protein kinase dsk1 with cell cycle stage dependent phosphorylation and localization Mol Biol Cell 4, 247–260 Ding JH, Zhong XY, Hagopian JC, Cruz MM, Ghosh G, Feramisco J, Adams JA & Fu XD (2006) Regulated cellular partitioning of SR protein-specific kinases in mammalian cells Mol Biol Cell 17, 876–885 Tang Z, Yanagida M & Lin RJ (1998) Fission yeast mitotic regulator Dsk1 is an SR protein-specific kinase J Biol Chem 273, 5963–5969 Tang Z, Kaufer NF & Lin RJ (2002) Interactions between two fission yeast serine ⁄ arginine-rich proteins and their modulation by phosphorylation Biochem J 368, 527–534 Tang Z, Tsurumi A, Alaei S, Wilson C, Chiu C, Oya J & Ngo B (2007) Dsk1p kinase phosphorylates SR proteins and regulates their cellular localization in fission yeast Biochem J 405, 21–30 FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS 581 Serine-arginine protein kinases T Giannakouros et al Kuroyanagi N, Onogi H, Wakabayashi T & Hagiwara M (1998) Novel SR-protein-specific kinase, SRPK2, disassembles nuclear speckles Biochem Biophys Res Commun 242, 357–364 Wang HY, Lin W, Dyck JA, Yeakley JM, Songyang Z, Cantley LC & Fu XD (1998) SRPK2: a differentially expressed SR protein-specific kinase involved in mediating the interaction and localization of pre-mRNA splicing factors in mammalian cells J Cell Biol 140, 737–750 10 Jang SW, Yang SJ, Ehlen A, Dong S, Khoury H, Chen J, Persson JL & Ye K (2008) Serine ⁄ arginine protein-specific kinase promotes leukemia cell proliferation by phosphorylating acinus and regulating cyclin A1 Cancer Res 68, 4559–4570 11 Siebel CW, Feng L, Guthrie C & Fu XD (1999) Conservation in budding yeast of a kinase specific for SR splicing factors Proc Natl Acad Sci USA 96, 5440–5445 12 Yeakley JM, Tronchere H, Olesen J, Dyck JA, Wang HY & Fu XD (1999) Phosphorylation regulates in vivo interaction and molecular targeting of serine ⁄ argininerich pre-mRNA splicing factors J Cell Biol 145, 447– 455 13 Yun CY & Fu XD (2000) Conserved SR protein kinase functions in nuclear import and its action is counteracted by arginine methylation in Saccharomyces cerevisiae J Cell Biol 150, 707–718 14 Kuroyanagi H, Kimura T, Wada K, Hisamoto N, Matsumoto K & Hagiwara M (2000) SPK-1, a C elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development Mech Dev 99, 51–64 15 Papoutsopoulou S, Nikolakaki E, Chalepakis G, Kruft V, Chevaillier P & Giannakouros T (1999) SR protein-specific kinase is highly expressed in testis and phosphorylates protamine Nucleic Acids Res 27, 2972–2980 16 Nikolakaki E, Kohen R, Hartmann AM, Stamm S, Georgatsou E & Giannakouros T (2001) Cloning and characterization of an alternatively spliced form of SR protein kinase that interacts specifically with scaffold attachment factor-B J Biol Chem 276, 40175–40182 17 Tsianou D, Nikolakaki E, Tzitzira A, Bonanou S, Giannakouros T & Georgatsou E (2009) The enzymatic activity of SR protein kinases and 1a is negatively affected by interaction with scaffold attachment factors B1 and FEBS J 276, 5212–5227 18 Nakagawa O, Arnold M, Nakagawa M, Hamada H, Shelton JM, Kusano H, Harris TM, Childs G, Campbell KP, Richardson JA et al (2005) Centronuclear myopathy in mice lacking a novel muscle-specific protein kinase transcriptionally regulated by MEF2 Genes Dev 19, 2066–2077 19 Xu Y, Yu W, Xiong Y, Xie H, Ren Z, Xu D, Lei M, Zuo B & Feng X (2010) Molecular characterization 582 20 21 22 23 24 25 26 27 28 29 30 and expression patterns of serine ⁄ arginine-rich specific kinase (SPRK3) in porcine skeletal muscle Mol Biol Rep doi:10.1007/s11033-010-9952-1 Johnson EL III, Fetter RD & Davis GW (2009) Negative regulation of active zone assembly by a newly identified SR protein kinase PLoS Biol 7, e1000193 Nieratschker V, Schubert A, Jauch M, Bock N, Bucher D, Dippacher S, Krohne G, Asan E, Buchner S & Buchner E (2009) Bruchpilot in ribbon-like axonal agglomerates, behavioral defects, and early death in SRPK79D kinase mutants of Drosophila PLoS Genet 5, e1000700 Portal D, Lobo GS, Kadener S, Prasad J, Espinosa JM, Pereira CA, Tang Z, Lin RJ, Manley JL, Kornblihtt AR et al (2003) Trypanosoma cruzi TcSRPK, the first protozoan member of the SRPK family, is biochemically and functionally conserved with metazoan SR protein-specific kinases Mol Biochem Parasitol 127, 9– 21 Liu S, Kang K, Zhang J, Ouyang Q, Zhou Z, Tian S & Xing M (2009) A novel Physarum polycephalum SR protein kinase specifically phosphorylates the RS domain of the human SR protein, ASF ⁄ SF2 Acta Biochim Biophys Sin (Shanghai) 41, 657–667 Wang HY, Arden KC, Bermingham JR Jr, Viars CS, Lin W, Boyer AD & Fu XD (1999) Localization of serine kinases, SRPK1 (SFRSK1) and SRPK2 (SFRSK2), specific for the SR family of splicing factors in mouse and human chromosomes Genomics 57, 310– 315 Lim LP & Burge CB (2001) A computational analysis of sequence features involved in recognition of short introns Proc Natl Acad Sci USA 98, 11193–11198 Colwill K, Feng LL, Yeakley JM, Gish GD, Caceres JF, Pawson T & Fu XD (1996) SRPK1 and Clk ⁄ Sty protein kinases show distinct substrate specificities for serine ⁄ arginine-rich splicing factors J Biol Chem 271, 24569–24575 Wang J, Xiao SH & Manley JL (1998) Genetic analysis of the SR protein ASF ⁄ SF2: interchangeability of RS domains and negative control of splicing Genes Dev 12, 2222–2233 Huang CJ, Tang Z, Lin RJ & Tucker PW (2007) Phosphorylation by SR kinases regulates the binding of PTB-associated splicing factor (PSF) to the pre-mRNA polypyrimidine tract FEBS Lett 581, 223–232 Papoutsopoulou S, Nikolakaki E & Giannakouros T (1999) SRPK1 and LBR protein kinases show identical substrate specificities Biochem Biophys Res Commun 255, 602–607 Calarco JA, Superina S, O’Hanlon D, Gabut M, Raj B, Pan Q, Skalska U, Clarke L, Gelinas D, van der Kooy D et al (2009) Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein Cell 138, 898–910 FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS T Giannakouros et al 31 Roscigno RF & Garcia-Blanco MA (1995) SR proteins escort the U4 ⁄ U6.U5 tri-snRNP to the spliceosome RNA 1, 692–706 32 Xiao SH & Manley JL (1997) Phosphorylation of the ASF ⁄ SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing Genes Dev 11, 334–344 33 Cao W, Jamison SF & Garcia-Blanco MA (1997) Both phosphorylation and dephosphorylation of ASF ⁄ SF2 are required for pre-mRNA splicing in vitro RNA 3, 1456–1467 34 Nikolakaki E, Du C, Lai J, Giannakouros T, Cantley L & Rabinow L (2002) Phosphorylation by LAMMER protein kinases: determination of a consensus site, identification of in vitro substrates, and implications for substrate preferences Biochemistry 41, 2055–2066 35 Labourier E, Rossi F, Gallouzi IE, Allemand E, Divita G & Tazi J (1998) Interaction between the N-terminal domain of human DNA topoisomerase I and the arginine-serine domain of its substrate determines phosphorylation of SF2 ⁄ ASF splicing factor Nucleic Acids Res 26, 2955–2962 36 Blaustein M, Pelisch F, Tanos T, Munoz MJ, Wengier D, Quadrana L, Sanford JR, Muschietti JP, Kornblihtt AR, Caceres JF et al (2005) Concerted regulation of nuclear and cytoplasmic activities of SR proteins by Akt Nat Struct Mol Biol 12, 1037–1044 37 Koizumi J, Okamoto Y, Onogi H, Mayeda A, Krainer AR & Hagiwara M (1999) The subcellular localization of SF2 ⁄ ASF is regulated by direct interaction with SR protein kinases (SRPKs) J Biol Chem 274, 11125–11131 38 Gilbert W, Siebel CW & Guthrie C (2001) Phosphorylation by Sky1p promotes Npl3p shuttling and mRNA dissociation RNA 7, 302–313 39 Lai MC, Lin RI & Tarn WY (2001) Transportin-SR2 mediates nuclear import of phosphorylated SR proteins Proc Natl Acad Sci USA 98, 10154–10159 40 Ngo JC, Chakrabarti S, Ding JH, Velazquez-Dones A, Nolen B, Aubol BE, Adams JA, Fu XD & Ghosh G (2005) Interplay between SRPK and Clk ⁄ Sty kinases in phosphorylation of the splicing factor ASF ⁄ SF2 is regulated by a docking motif in ASF ⁄ SF2 Mol Cell 20, 77–89 41 Hamelberg D, Shen T & McCammon JA (2007) A proposed signaling motif for nuclear import in mRNA processing via the formation of arginine claw Proc Natl Acad Sci USA 104, 14947–14951 42 Dagher SF & Fu XD (2001) Evidence for a role of Sky1p-mediated phosphorylation in 3¢ splice site recognition involving both Prp8 and Prp17 ⁄ Slu4 RNA 7, 1284–1297 43 Kamachi M, Le TM, Kim SJ, Geiger ME, Anderson P & Utz PJ (2002) Human autoimmune sera as molecular Serine-arginine protein kinases 44 45 46 47 48 49 50 51 52 53 54 55 FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS probes for the identification of an autoantigen kinase signaling pathway J Exp Med 196, 1213–1225 Mathew R, Hartmuth K, Mohlmann S, Urlaub H, Ficner R & Luhrmann R (2008) Phosphorylation of human PRP28 by SRPK2 is required for integration of the U4 ⁄ U6-U5 tri-snRNP into the spliceosome Nat Struct Mol Biol 15, 435–443 Hayes GM, Carrigan PE & Miller LJ (2007) Serinearginine protein kinase overexpression is associated with tumorigenic imbalance in mitogen-activated protein kinase pathways in breast, colonic, and pancreatic carcinomas Cancer Res 67, 2072–2080 Zhong XY, Ding JH, Adams JA, Ghosh G & Fu XD (2009) Regulation of SR protein phosphorylation and alternative splicing by modulating kinetic interactions of SRPK1 with molecular chaperones Genes Dev 23, 482–495 Jang SW, Liu X, Fu H, Rees H, Yepes M, Levey A & Ye K (2009) Interaction of Akt-phosphorylated SRPK2 with 14-3-3 mediates cell cycle and cell death in neurons J Biol Chem 284, 24512–24525 Zhang Z & Krainer AR (2004) Involvement of SR proteins in mRNA surveillance Mol Cell 16, 597–607 ´ Michlewski G, Sanford JR & Caceres JF (2008) The splicing factor SF2 ⁄ ASF regulates translation initiation by enhancing phosphorylation of 4E-BP1 Mol Cell 30, 179–189 Windgassen M, Sturm D, Cajigas IJ, Gonzalez CI, Seedorf M, Bastians H & Krebber H (2004) Yeast shuttling SR proteins Npl3p, Gbp2p, and Hrb1p are part of the translating mRNPs, and Npl3p can function as a translational repressor Mol Cell Biol 24, 10479–10491 Sciabica KS, Dai QJ & Sandri-Goldin RM (2003) ICP27 interacts with SRPK1 to mediate HSV splicing inhibition by altering SR protein phosphorylation EMBO J 22, 1608–1619 Bell I, Martin A & Roberts S (2007) The E1^E4 protein of human papillomavirus interacts with the serine-arginine-specific protein kinase SRPK1 J Virol 81, 5437–5448 Daub H, Blencke S, Habenberger P, Kurtenbach A, Dennenmoser J, Wissing J, Ullrich A & Cotten M (2002) Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein J Virol 76, 8124–8137 Zheng Y, Fu XD & Ou JH (2005) Suppression of hepatitis B virus replication by SRPK1 and SRPK2 via a pathway independent of the phosphorylation of the viral core protein Virology 342, 150–158 Fukuhara T, Hosoya T, Shimizu S, Sumi K, Oshiro T, Yoshinaka Y, Suzuki M, Yamamoto N, Herzenberg LA & Hagiwara M (2006) Utilization of host SR protein kinases and RNA-splicing machinery during viral replication Proc Natl Acad Sci USA 103, 11329–11333 583 Serine-arginine protein kinases T Giannakouros et al 56 Hayes GM, Carrigan PE, Beck AM & Miller LJ (2006) Targeting the RNA splicing machinery as a novel treatment strategy for pancreatic carcinoma Cancer Res 66, 3819–3827 57 Hishizawa M, Imada K, Sakai T, Ueda M, Hori T & Uchiyama T (2005) Serological identification of adult T-cell leukaemia-associated antigens Br J Haematol 130, 382–390 58 Schenk PW, Boersma AW, Brandsma JA, den Dulk H, Burger H, Stoter G, Brouwer J & Nooter K (2001) SKY1 is involved in cisplatin-induced cell kill in Saccharomyces cerevisiae, and inactivation of its human homologue, SRPK1, induces cisplatin resistance in a human ovarian carcinoma cell line Cancer Res 61, 6982–6986 59 Schenk PW, Stoop H, Bokemeyer C, Mayer F, Stoter G, Oosterhuis JW, Wiemer E, Looijenga LH & Nooter K (2004) Resistance to platinum-containing chemotherapy in testicular germ cell tumors is associated with downregulation of the protein kinase SRPK1 Neoplasia 6, 297–301 60 Zahler AM, Neugebauer KM, Lane WS & Roth MB (1993) Distinct functions of SR proteins in alternative pre-mRNA splicing Science 260, 219–222 61 Fu X-D (1995) The superfamily of arginine ⁄ serine-rich splicing factors RNA 1, 663–680 62 Chirat F, Arkhis A, Martinage A, Jaquinot M, Che` vaillier P & Sautiere P (1993) Phosphorylation of human sperm protamines HP1 and HP2: identification of phosphorylation sites Biochim Biophys Acta 1203, 109–114 63 Balhorn R (2007) The protamine family of sperm nuclear proteins Genome Biol 8, 227 64 Sassone-Corsi P (2002) Unique chromatin remodeling and transcriptional regulation in spermatogenesis Science 296, 2176–2178 65 Oliva R & Dixon GH (1991) Vertebrate protamine genes and the histone-to-protamine replacement reaction Prog Nucleic Acid Res Mol Biol 40, 25–94 66 Wu JY, Ribar TJ, Cummings DE, Burton KA, McKnight GS & Means AR (2000) Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4 Nat Genet 25, 448– 452 67 Mylonis I, Drosou V, Brancorsini S, Nikolakaki E, Sassone-Corsi P & Giannakouros T (2004) Temporal association of protamine with the inner nuclear membrane protein lamin B receptor during spermiogenesis J Biol Chem 279, 11626–11631 ´ 68 Valcarcel J & Green MR (1996) The SR protein family: pleiotropic functions in pre-mRNA splicing Trends Biochem Sci 21, 296–301 69 Biggiogera M, Muller S, Courtens JL, Fakan S & Romanini MG (1992) Immunoelectron microscopical distribution of histones H2B and H3 and protamines 584 70 71 72 73 74 75 76 77 78 79 80 81 in the course of mouse spermiogenesis Microsc Res Tech 20, 259–267 Cheung P, Allis CD & Sassone-Corsi P (2000) Signaling to chromatin through histone modifications Cell 103, 263–271 Jakel S, Albig W, Kutay U, Bischoff FR, Schwamborn K, ¨ Doenecke D & Gorlich D (1999) The importin ¨ beta ⁄ importin heterodimer is a functional nuclear import receptor for histone H1 EMBO J 18, 2411– 2423 Pradeepa MM, Manjunatha S, Sathish V, Agrawal S & Rao MR (2008) Involvement of importin-4 in the transport of transition protein into the spermatid nucleus Mol Cell Biol 28, 4331–4341 Erez O & Kahana C (2001) Screening for modulators of spermine tolerance identifies Sky1, the SR protein kinase of Saccharomyces cerevisiae, as a regulator of polyamine transport and ion homeostasis Mol Cell Biol 21, 175–184 Georgatos SD (2001) The inner nuclear membrane: simple, or very complex? EMBO J 20, 2989–2994 Pyrpasopoulou A, Meier J, Maison C, Simos G & Georgatos SD (1996) The lamin B receptor (LBR) provides essential chromatin docking sites at the nuclear envelope EMBO J 15, 7108–7119 Polioudaki H, Kourmouli N, Drosou V, Bakou A, Theodoropoulos PA, Singh PB, Giannakouros T & Georgatos SD (2001) Histones H3 ⁄ H4 form a tight complex with the inner nuclear membrane protein LBR and heterochromatin protein EMBO Rep 2, 920– 925 Makatsori D, Kourmouli N, Polioudaki H, Shultz LD, McLean K, Theodoropoulos PA, Singh PB & Georgatos SD (2004) The inner nuclear membrane protein lamin B receptor forms distinct microdomains and links epigenetically marked chromatin to the nuclear envelope J Biol Chem 279, 25567–25573 Ye Q & Worman HJ (1994) Primary structure analysis and lamin B and DNA binding of human LBR, an integral protein of the nuclear envelope inner membrane J Biol Chem 269, 11306–11311 Nikolakaki E, Drosou V, Sanidas I, Peidis P, Papamarcaki T, Iakoucheva LM & Giannakouros T (2008) RNA association or phosphorylation of the RS domain prevents aggregation of RS domain-containing proteins Biochim Biophys Acta 80, 214–225 Nikolakaki E, Meier J, Simos G, Georgatos SD & Giannakouros T (1997) Mitotic phosphorylation of the lamin B receptor by a serine ⁄ arginine kinase and p34(cdc2) J Biol Chem 272, 6208–6213 Takano M, Koyama Y, Ito H, Hoshino S, Onogi H, Hagiwara M, Furukawa K & Horigome T (2004) Regulation of binding of lamin B receptor to chromatin by SR protein kinase and cdc2 kinase in Xenopus egg extracts J Biol Chem 279, 13265–13271 FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS T Giannakouros et al 82 Loomis RJ, Naoe Y, Parker JB, Savic V, Bozovsky MR, Macfarlan T, Manley JL & Chakravarti D (2009) Chromatin binding of SRp20 and ASF ⁄ SF2 and dissociation from mitotic chromosomes is modulated by histone H3 serine 10 phosphorylation Mol Cell 33, 450–461 83 Misteli T (2005) Concepts in nuclear architecture Bioessays 27, 477–487 84 Andrulis ED, Neiman AM, Zappulla DC & Sternglanz R (1998) Perinuclear localization of chromatin facilitates transcriptional silencing Nature 394, 592– 595 85 Wolgemuth DJ, Lele KM, Jobanputra V & Salazar G (2004) The A-type cyclins and the meiotic cell cycle in mammalian male germ cells Int J Androl 27, 192– 199 86 Yang R, Nakamaki T, Lubbert M, Said J, Sakashita A, Freyaldenhoven BS, Spira S, Huynh V, Muller C & ă Koefer HP (1999) Cyclin A1 expression in leukemia and normal hematopoietic cells Blood 93, 2067–2074 87 Sahara S, Aoto M, Eguchi Y, Imamoto N, Yoneda Y & Tsujimoto Y (1999) Acinus is a caspase-3-activated protein required for apoptotic chromatin condensation Nature 401, 168–173 88 Schwerk C, Prasad J, Degenhardt K, ErdjumentBromage H, White E, Tempst P, Kidd VJ, Manley JL, Lahti JM & Reinberg D (2003) ASAP, a novel protein complex involved in RNA processing and apoptosis Mol Cell Biol 23, 2981–2990 89 Kim JK & Diehl JA (2009) Nuclear cyclin D1: an oncogenic driver in human cancer J Cell Physiol 220, 292–296 90 Xiao R, Sun Y, Ding JH, Lin S, Rose DW, Rosenfeld MG, Fu X-D & Li X (2007) Splicing regulator SC35 is essential for genomic stability and cell proliferation during mammalian organogenesis Mol Cell Biol 27, 5393–5402 91 Lin S, Coutinho-Mansfield G, Wang D, Pandit S & Fu XD (2008) The splicing factor SC35 has an active role in transcriptional elongation Nat Struct Mol Biol 15, 819–826 92 Rocha S, Martin AM, Meek DW & Perkins ND (2003) p53 represses cyclin D1 transcription through down regulation of Bcl-3 and inducing increased association of the p52 NF-kappaB subunit with histone deacetylase Mol Cell Biol 23, 4713–4727 93 Lin J, Handschin C & Spiegelman BM (2005) Metabolic control through the PGC-1 family of transcription coactivators Cell Metab 6, 361–370 94 Puigserver P, Wu Z, Park CW, Graves R, Wright M & Spiegelman BM (1998) A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis Cell 92, 829–839 ´ 95 Canto C & Auwerx J (2010) Clking on PGC-1alpha to inhibit gluconeogenesis Cell Metab 11, 6–7 Serine-arginine protein kinases 96 Puigserver P, Rhee J, Donovan J, Walkey CJ, Yoon JC, Oriente F, Kitamura Y, Altomonte J, Dong H, Accili D et al (2003) Insulin-regulated hepatic gluconeogenesis through FOXO1-PGC-1alpha interaction Nature 423, 550–555 97 Li X, Monks B, Ge Q & Birnbaum MJ (2007) Akt ⁄ PKB regulates hepatic metabolism by directly inhibiting PGC-1alpha transcription coactivator Nature 447, 1012–1016 98 Rodgers JT, Haas W, Gygi SP & Puigserver P (2010) Cdc2-like kinase is an insulin-regulated suppressor of hepatic gluconeogenesis Cell Metab 11, 23–34 99 Rodgers JT, Lerin C, Gerhart-Hines Z & Puigserver P (2008) Metabolic adaptations through the PGC-1 alpha and SIRT1 pathways FEBS Lett 582, 46–53 100 Fogal V, Richardson AD, Karmali PP, Scheffler IE, Smith JW & Ruoslahti E (2010) Mitochondrial p32 protein is a critical regulator of tumor metabolism via maintenance of oxidative phosphorylation Mol Cell Biol 30, 1303–1318 101 Muta T, Kang D, Kitajima S, Fujiwara T & Hamasaki N (1997) p32 protein, a splicing factor 2-associated protein, is localized in mitochondrial matrix and is functionally important in maintaining oxidative phosphorylation J Biol Chem 272, 24363–24370 102 Soltys BJ, Kang D & Gupta RS (2000) Localization of P32 protein (gC1q-R) in mitochondria and at specific extramitochondrial locations in normal tissues Histochem Cell Biol 114, 245–255 103 Nikolakaki E, Simos G, Georgatos SD & Giannakouros T (1996) A nuclear envelope-associated kinase phosphorylates arginine-serine motifs and modulates interactions between the lamin B receptor and other nuclear proteins J Biol Chem 271, 8365–8372 104 Petersen-Mahrt SK, Estmer C, Ohrmalm C, Matthews DA, Russell WC & Akusjarvi G (1999) The splicing ă factor-associated protein, p32, regulates RNA splicing by inhibiting ASF ⁄ SF2 RNA binding and phosphorylation EMBO J 18, 1014–1024 105 Cao W & Garcia-Blanco MA (1998) A serine ⁄ argininerich domain in the human U1 70k protein is necessary and sufficient for ASF ⁄ SF2 binding J Biol Chem 273, 20629–20635 106 Yue BG, Ajuh P, Akusjarvi G, Lamond AI & Kreivi JP (2000) Functional coexpression of serine protein kinase SRPK1 and its substrate ASF ⁄ SF2 in Escherichia coli Nucleic Acids Res 28, E14 107 Nolen B, Yun CY, Wong CF, McCammon JA, Fu XD & Ghosh G (2001) The structure of Sky1p reveals a novel mechanism for constitutive activity Nat Struct Biol 8, 176–183 108 Ngo JC, Gullingsrud J, Giang K, Yeh MJ, Fu XD, Adams JA, McCammon JA & Ghosh G (2007) SR protein kinase is resilient to inactivation Structure 15, 123–133 FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS 585 Serine-arginine protein kinases T Giannakouros et al 109 Mylonis I & Giannakouros T (2003) Protein kinase CK2 phosphorylates and activates the SR proteinspecific kinase Biochem Biophys Res Commun 301, 650–656 110 Fukuda M, Asano S, Nakamura T, Adachi M, Yoshida M, Yanagida M & Nishida E (1997) CRM1 is responsible for intracellular transport mediated by the nuclear export signal Nature 390, 308–311 111 Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P & Mann M (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks Cell 127, 635–648 112 Dephoure N, Zhou C, Villen J, Beausoleil SA, Bakalarski CE, Elledge SJ & Gygi SP (2008) A quantitative atlas of mitotic phosphorylation Proc Natl Acad Sci USA 105, 10762–10767 586 113 Daub H, Olsen JV, Bairlein M, Gnad F, Oppermann FS, Korner R, Greff Z, Keri G, Stemmann O & Mann M (2008) Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle Mol Cell 31, 438–448 114 Chiodi I, Biggiogera M, Denegri M, Corioni M, Weighardt F, Cobianchi F, Riva S & Biamonti G (2000) Structure and dynamics of hnRNP-labelled nuclear bodies induced by stress treatments J Cell Sci 113 (Pt 22), 4043–4053 115 Denegri M, Chiodi I, Corioni M, Cobianchi F, Riva S & Biamonti G (2001) Stress-induced nuclear bodies are sites of accumulation of pre-mRNA processing factors Mol Biol Cell 12, 3502–3514 FEBS Journal 278 (2011) 570–586 ª 2011 The Authors Journal compilation ª 2011 FEBS ... kinases and 1a is negatively affected by interaction with scaffold attachment factors B1 and FEBS J 276, 5212–5227 18 Nakagawa O, Arnold M, Nakagawa M, Hamada H, Shelton JM, Kusano H, Harris TM,... cerevisiae and S pombe with one gene (Sky1 and Dsk1, respectively); Candida albicans with two (QSAA48 and QS9Q27); Aspergilus niger with nine (A2 QAE4, A2 QB94, A2 QC46, A5 AB23, A2 QWQ2, A2 QX01, A2 QX98,... Nikolakaki E, Kohen R, Hartmann AM, Stamm S, Georgatsou E & Giannakouros T (2001) Cloning and characterization of an alternatively spliced form of SR protein kinase that interacts specifically with