A NOVEL INTEGRAL MEMBRANE POOL OF PROTEIN KINASE C AND ITS ROLE IN MAMMALIAN CELL SIGNALING ZHU YIMIN (M.B.B.S, and M. Med) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2004 Chapter Introduction 1.1 The organization of signal transduction pathways 1.1.1 Extracellular signals According to the nature of the molecules, extracellular signals include proteins, small peptides, amino acids, nucleotides, steroids, retinoids, and fatty acid derivatives and dissolved gases such as nitric oxide and carbon monoxide. These signals interact with specific proteins called receptors in target cells and initiate a biological response in the target cell. According to their subcellular localization, receptors are divided into two groups: cell surface receptors and intracellular receptors. Often, the signaling cell and target cell are of different cell types. Sometimes cells can also send signals to themselves and other cells of the same type. Extracellular signals can act on target cells over either short or long distance. There are several different modes of signaling. In contact-dependent signaling, signals are exposed to the extracellular space while still tightly bound to the signaling cell’s surface. In paracrine signaling, signals are secreted from the signaling cell and diffuse in the immediate environment of the signaling cell. In synaptic signaling, when the dendrites of neurons receive signals, the neuron sends electrical impulses rapidly along the axon of the neuron to the end of the axon to elicit the release of neurotransmitters into the synapse and the signals are finally delivered to the postsynaptic target cell. In endocrine signaling, the endocrine cells secrete signal molecules (hormones) to the blood stream that are delivered to different parts of the multicellular organism. Some extracellular signals can act on themselves or other cells of the same type. In autocrine signaling, the cells that secrete the signals and the cells that accept the signals are identical. Gap junctions are specific cell-to-cell junctions that can connect the cytoplasms of adjacent cells via water-filled channels. 1.1.2 Cell signaling machinery 1.1.2.1 Receptors of the signals The receptors of signals can localize in different compartments in cells and the same receptors situated in different locale may have different biological functions. 1.1.2.1.1 Cell surface receptors Hydrophilic signal molecules must bind to receptors on the cell surface, which transduce the signals. Most cell surface receptors belong to one of three classes of receptors: ion-channellinked receptors, G-protein-linked receptors, and enzyme-linked receptors1. 1.1.2.1.1.1. Ion-channel-linked receptors Ion-channel-linked receptors are involved in synaptic signaling. The receptors for some transmitters themselves are ion channels. Alternatively, the receptors can link with isolated ion channels. When the transmitters reach the receptors in the postsynaptic membrane, the conformation of ion-channel/receptors changes, which in turn influences the flowing of ions such as Ca2+, Na+, or K+ in the postsynaptic membrane. If the receptors are not ion channels, the conformational change of the receptor can affect the conformation of the linked ion channel, resulting in alteration of the flow of ions. With the change of flowing of ions, the potential of target cells subsequently changes, so the signals can be transduced by ionchannel-linked receptors2. 1.1.2.1.1.2 G-protein-linked receptors All of the G-protein-linked receptors belong to a large family of homologous, seven-pass transmembrane proteins. G-protein-linked receptors indirectly regulate the activity of a separate protein which is also membrane-bound. The intermediate of the regulation is a third protein, called a trimeric GTP-binding protein (G-protein). The membrane-bound proteins are either ion channels or enzymes. When the signals bind to the receptors in the target cells and activate the target proteins, the conformation of the enzymes or the permeability of the ion channels are changed. Subsequently, signaling proteins in cells can relay the signals to different compartments in cells3,4. 1.1.2.1.1.3 Enzyme-linked receptors There are five classes of enzyme-linked receptors; (1) receptor tyrosine kinases, (2) tyrosine kinase-associated receptors, (3) receptor serine/threonine kinases, (4) transmembrane guanylyl cyclases, and (5) histidine-kinase-associated receptors. The first two classes are the most abundant in cells. Enzyme-linked receptors are single-pass transmembrane proteins with an extracellular ligand-binding domain and an intracellular catalytic or enzyme-binding domain. The great majority of the receptors are themselves protein kinases or are associated with kinases. Binding of agonists to receptors induces a conformational change of the receptors. Such conformational change leads to the activation of kinases that are either intrinsic to the receptor or associated with the receptor. Thus the signals are transduced from the extracellular to the intracellular environment5,6. Besides the three groups of cell surface receptors, there are some other cell surface receptors that not fit into the three classes. There is one group of cell surface receptors that activate the signaling pathways depending on proteolysis. For example, the receptor protein Notch is activated by cleavage. In vertebrates, the majority of the Notch protein is targeted to the cell surface after processing by a furin-like convertase (FLC) at cleavage site 17,8. The S1 cleavage divides Notch into two polypeptides: one contains almost the entire extracellular domain and the other contains a short ectodomain stub followed by a transmembrane domain and a long cytoplasmic tail. After ligand binding, a change occurs that renders the region just proximal to the membrane susceptible to cleavage by metalloproteases of the ADAM family (a disintegrin and metalloprotease), especially at a position 12 amino acids from the membrane. This site is referred to as the S2 site. After ligand-induced ectodomain shedding, Notch undergoes cleavage at a third site (S3) located within the transmembrane domain by an unusual enzyme called γ-secretase. The freed intracellular domain enters the nucleus, where it switches a DNA-bound corepressor complex into an activating complex, leading to activation of selected target genes 9,10. 1.1.2.1.2 Nuclear receptors Hydrophobic signaling molecules can diffuse directly across the plasma membrane of target cells and bind to their intracellular receptors. These agonists include steroid hormones, thyroid hormones, retinoids, and vitamin D. Upon binding to the receptors, they activate the receptors that can bind to specific DNA sequences adjacent to the target genes to regulate the transcription of such genes. The receptors are structurally related and are part of the nuclear receptor family. Some receptors that are activated by intracellular metabolites are also included in this family. Some nuclear receptors, such as those for cortisol, are located in the cytoplasm, and translocate to the nucleus after ligand binding, while other nuclear receptors, those for thyroid hormone and retinoids, are bound to DNA even in the absence of ligands. After the binding of hormones to the receptors, the inhibitory protein complexes bound to the inactive receptors dissociate and the receptors bound to coactivator proteins induce gene transcription11. 1.1.2.1.3 Intracellular enzymes as receptors Nitric oxide gas (NO) and carbon monoxide (CO) can rapidly diffuse across the membrane and bind to iron in the active form of guanylyl cyclase, which stimulates this enzyme to produce the small intracellular mediator cyclic GMP. The cyclic GMP can induce responses in target cells, for example, keeping blood vessels relaxed12. 1.1.2.2 Intracellular signaling molecules After extracellular signaling molecules bind to the receptors, the signals are relayed into the cell interior by a combination of small and large intracellular signaling molecules. The former are second messengers; the latter are called intracellular signaling proteins including G proteins, small GTPase and protein kinases13-15. 1.2 Protein kinases 1.2.1 History and Definition The presence of phosphorus in proteins has been known since the early part of the last century. The function of phosphoproteins was considered to be mainly nutritional16. In the early 1940s, Cori and his colleagues showed that the enzyme glycogen phosphorylase exists in two forms: an inactive form called phosphorylase b and an active form termed phosphorylase a. These two forms of the enzyme are interconvertible in intact cells17,18. It was not until the 1950s that the mechanism of this interconversion was discovered when two independent groups reported that enzymes of phosphorylation-dephosphorylation were involved19,20. Based on their discoveries, the inactive phosphorylase b can be activated by phosphorylation catalyzed by a newly isolated enzyme, phosphorylase kinase (a protein kinase). Phosphorylase a can be inactivated by dephosphorylation catalyzed by a specific phosphatase. Many kinases have been discovered in the past 50 years. It is now universally accepted that the reversible phosphorylation of proteins regulates most aspects of cell life21-26. Protein kinases and protein phosphatases provide the basis of a network that allows extracellular signals to coordinate biochemical functions within the target cells27-30. It is therefore not surprising that a large number of protein kinases exist. It seems likely that in mammalian cells over 500 different kinase molecules have evolved to form the requisite signal transducing networks31. In contrast, a limited number of protein phosphatases are present in the cell. With the full appreciation that protein kinases and protein phosphatases are equally important in signal transduction and integration within the cells, however, this research focuses on protein kinases. Protein kinases are defined as enzymes that transfer a phosphoryl group from a phosphate donor onto an acceptor amino acid in a substrate protein. Most kinases use ATP as the phosphoryl group donor although several kinases also utilize GTP. The protein kinases, which catalyze the phosphorylation step in various phosphorylation-dephosphorylation systems, can be divided into two main classes, the serine-threonine protein kinases and the tyrosine protein kinases based on the phosphorylated residues in the substrate protein. Each class can be subdivided into groups or entities depending on the nature of the agents that regulate activity. 1.2.2 Classification and superfamily of protein kinases The eukaryotic protein kinases make up a large superfamily of homologous proteins. The kinase domains that define this group of enzymes contain 12 conserved subdomains that fold into a common catalytic core structure32. In the mid’s of 1990, phylogenetic trees derived from an alignment of kinase domain amino acid sequences served as the basis for the classification by Hanks and Hunter. Protein kinases can be divided into groups: (1) the AGC group; (2) the CaMK group; (3) the CMGC group; (4) conventional protein tyrosine kinase (RTK) group. Recently, the near completion of the human genome sequence allows the identification of almost all human protein kinases32. The classification of protein kinases has been extended from the Hanks and Hunter’s four broad groups, 44 families and 51 subfamilies to adding four new groups including the STE group, CK1 group, TTBK group and TKL group encompassing 90 families and 145 subfamilies31. In humans, a total of 518 protein kinases have been identified including 478 human eukaryotic protein kinases and 40 atypical protein kinases. The protein kinases constitute about 1.7 % of all human genes31. According to the type of amino acid phosphorylated, the protein kinases can be divided into two subgroups: protein serine/threonine kinases and protein tyrosine kinases. Serine/threonine protein kinases were the first discovered kinases that catalyze the phosphorylation of hydroxyamino acids. All of the known protein kinases including tyrosine kinases share a related catalytic domain of about 260 amino acids and are distinguished by their unique regulatory domains32. Tyrosine kinases are the other major kinases in the protein kinase family. Protein tyrosine phosphorylation, first encountered in 1979 as an activity of viral transforming gene products33, was quickly recognized to play an important role in transducing growth factor receptor signals across the plasma membrane. With time it has become clear that tyrosine phosphorylation also regulates protein function in many other cellular processes including the cell cycle, transcription, and synaptic transmission. Tyrosine kinases also have a catalytic domain of about 260 amino acids just like serine/threonine kinases. A number of residues within this domain are highly conserved between both types of protein kinases, but the tyrosine kinases and serine/threonine kinases are distinguished by specific signature motifs34. The constitution of major serine/threonine kinases and tyrosine kinases is shown in Figure1.1. Figure 1.1 Major members of the serine/threonine kinase family and tyrosine kinase family (adapted from Hanks S.K, et al.34). In addition to serine/threonine kinases and tyrosine kinases, there are reports on other types of protein kinases that phosphorylate proteins on histidine35,36 or on arginines37. 1.3 Protein kinase C superfamily The protein kinase C superfamily belongs to the AGC family of serine-threonine kinases38,39. PKC was originally discovered by Nishizuka and coworkers as a proteolytically activated kinase40,41. However, it was subsequently demonstrated that PKC was reversibly activated by a lipid-soluble, membrane-associated factor in the presence of Ca2+42,43. Diacylglycerol (DAG) was later shown to be the neutral lipid that activated PKC in a phosphatidylserine (PS)- and Ca2+-dependent manner44-46. This discovery linked PKC to major signal transduction pathways involving phosphatidylinositol turnover. PKC is now known to be an increasingly large family with multiple subspecies, which represents one of the major classes of downstream targets for lipid second messengers. PKCs are involved in the regulation of cell proliferation, differentiation, and immunity and are implicated in the development of diseases such as cancer, neurodegenerative diseases, heart attack and diabetes47. 1.3.1 PKC family members After 27 years of study, exhaustive genetic screening has defined a superfamily of mammalian PKC of at least 14 isozymes. The mammalian PKC isotypes have been grouped into subfamilies on the basis of their biochemical properties and sequence homology: (1) conventional PKCs (cPKC) containing α, βІ, βII, γ48; (2) novel PKCs (nPKC) including ε, η, δ, θ49; (3) atypical PKCs containing ι/λ, ζ (aPKC)50, and (4) protein kinase C-related kinases (PRKs) 1-3. Sequence comparisons place PKC isotypes into subgroups (Figure 1.2) including the newly discovered PKCζII which does not contain a catalytic domain51. These groupings are almost identical to the classification based on biochemical properties, with the only exception being the splitting of the novel PKCs into two pairs of very closely related kinases, namely δ, θ and ε, η. Closer examination of protein sequence alignments between PKC isotypes reveals the presence of blocks of homology between family members. These conserved regions confer a specific localization and/or activation input to different isozymes. Figure 1.2 Dendrogram based on a sequence comparison of the PKC superfamily. Protein sequences of the fully-cloned members of the human PKC superfamily were compared using Clustal V software with PAM 250 residue tables (adapted from Ponting, C.P. & Parker, P.J.52). 1.3.2 Structure of PKCs All PKC isozymes, except for PKCζII, consist of a single polypeptide chain with an Nterminal regulatory domain and a C-terminal catalytic domain interspersed by a hinge region (the V3 domain)50,53. The carboxyl-terminal domains of all these PKC molecules are the catalytic domains containing consensus sequences for ATP- and substrate-binding sites34,54. The amino-terminal domains of PKC are quite diverse and are referred to as regulatory viii 3.2 Exploration of mechanisms responsible for the integration of PRK1 into the membrane 82 3.2.1 PRK1 is not a classical integral membrane protein 82 3.2.2 Topology of PRK1 in the plasma membrane 83 3.2.3 The catalytic activity of PRK1 is not required for integration of PRK1 into the cellular membrane 85 3.2.4 The integration of PRK1 into membrane is independent of RhoA interaction 85 3.2.5 The integration of PRK1 into the cellular membrane is independent of internal myristoylation and palmitoylation 86 3.2.6 Phosphorylation of residue 377 is critical for the integration of PRK1 into cellular membranes 87 3.2.6.1 Generation of specific phosphor-Ser377 antibody 88 3.2.6.2 Specificity of phospho-377 antibody (P-P377) 89 3.2.6.3 Ser377-phosphorylated PRK1 is distributed throughout cells 89 3.2.6.4 Serine-377 is not an intramolecular autophosphorylated site 90 3.2.6.5 Characterization of PRK1-S377A mutant 91 3.3 Characterization of the integral membrane pool of PRK1 94 3.3.1 Activity of integral membrane pool of exogenous PRK1 in COS1 cells and endogenous PRK1 in rat erythrocytes 94 3.3.2 The Ser377-phosphorylated PRK1 in the plasma membrane of hepatocytes is dynamically regulated during liver regeneration 3.3.3 The integral membrane pool of PRK1 can be activated by RhoA 95 96 3.3.3.1 Lysophosphatidic acid (LPA) increases the activity of PRK1 in total cell lysate 3.3.3.2 PRK1 in the aqueous phase cannot be activated by LPA 96 97 ix 3.3.3.3 LPA dramatically activates membrane-bound PRK1 98 3.3.3.4 LPA fails to activate PRK1-S377A in cells 100 3.4 RhoA signals via an integral membrane pool of its effector PRK1 101 3.4.1 Neurite retraction induced by RhoA in mouse neuronal cells is mediated by the integral membrane pool of PRK1 102 3.4.2 RhoA-mediated and ligand-dependent transcriptional activation of androgen receptors is mediated by the integral membrane pool of PRK1105 3.4.3 The distribution patterns of WT-PRK1 and PRK1-S377A in HEK293 and N1E-115 cells are similar upon LPA stimulation 3.5 Conclusion and Discussion 106 107 3.5.1 Experimental findings for the integral membrane pool of PRK1 107 3.5.2 LPA activates only the integral membrane pool of PRK1 in cells 108 3.5.3 Biochemical and cell biological basis for using PRK1-S377A to study the function of the integral membrane pool of PRK1 3.5.4 Clinical significance of the integral membrane pool of PRK1 113 114 Chapter 4: Results Part 2: Signaling via integral membrane pool of PKC in Mammalian cells 117 4.1 Identification of an integral membrane pool of PKC in mammalian tissues and cells 4.1.1 Endogenous PKCs in NIH3T3 cells are integral membrane proteins 117 117 4.1.2 Ectopically expressed PKCs in COS1 cells are integral membrane proteins in addition to being cytosolic proteins 117 4.1.3 The integration of PKCs into the cellular membrane is tissue-specific 118 4.2 The mechanisms responsible for the integration of PKCα into membranes 119 x 4.2.1 The catalytic activity of PKCα is not required for the integration of PKCα into the cellular membrane 4.2.2 Topology of integral membrane PKCα 119 120 4.2.3 Internal myristoylation and palmitoylation are not responsible for the integration of PKCs into cellular membranes 4.2.4 C2-V3 domain is critical for membrane integration of PKCα 4.3 Translocation of PKCα by DAG or TPA 121 122 123 4.3.1 Short term TPA treatment increases the amount of ectopically expressed PKCα in both peripheral membrane pool and integral membrane pool 123 4.3.2 Short term TPA treatment increases the amount of endogenous PKCα in both peripheral membrane pool and integral membrane pool in NIH3T3 cells 125 4.3.3 DOG (sn-1, 2-dioctanoylglycerol) has a similar effect as TPA on the translocation of endogenous PKCα 4.4 The integral membrane pool of PKCα is functional 126 127 4.4.1 Ectopically expressed PKCα in the integral membrane pool is catalytically active 127 4.4.2 The specific activity of endogenous PKCα in the integral membrane pools of NIH3T3 cells 128 4.4.3 TPA treatment does not increase the specific activity of PKCα in the soluble fraction 129 4.4.4 TPA treatment slightly increases the specific activity of PKCα in the integral membrane pool 130 4.4.4.1 TPA treatment slightly increases the specific activity of ectopically expressed PKCα in the integral membrane pool 130 4.4.4.2 TPA treatment slightly increases the specific activity of endogenous xi PKCα and PKCε in the integral membrane pool of NIH3T3 cells 131 4.5 The physiological role of the integral membrane pool of PKCα in vivo 133 4.5.1 Characterization of PKCα-D294G and PKCα-E265G mutants 133 4.5.1.1 PKCα mutants translocate to the peripheral membrane upon short term TPA treatment 4.5.1.2 PKCα mutants have very similar basal activity as WT-PKCα 133 134 4.5.1.3 PKCα mutants have very similar specific activity as WT-PKCα in vitro upon DAG stimulation 135 4.5.1.4 PKCα mutants and WT-PKCα have a very similar subcellular distribution pattern in cells 136 4.5.2 MARCKS translocation upon TPA stimulation is mediated by the integral membrane pool of PKCα 137 4.5.3 Only wild-type PKCα but not the PKCα-D294G mutant is able to activate MAPK in vivo 142 4.5.4 Only wild-type PKCα but not the PKCα-D294G mutant is able to augment neurite outgrowth in neuronal cells in vivo 144 4.6 Conclusion and Discussion 145 Chapter 5: Discussion 153 5.1 Summary of our findings 153 5.2 How the integral membrane protein is defined 154 5.3 How specificity and efficiency of cell signaling are achieved 155 5.3.1 Signals are transduced by translocation based on diffusion and reversible interaction 5.3.2 Cytoarchitecture and physical properties of the cytoplasm 156 157 xii 5.3.2.1 The cell interior is crowded 157 5.3.2.2 The cytoplasm is a complicated solution 158 5.3.2.3 The cytoplasm is a heterogeneous solution 158 5.3.2.4 Diffusion of proteins and other macromolecules within the cytoplasm 159 5.3.3 Signals are transduced by movement of macromolecules depending on the rate of rebinding of different proteins 160 5.3.4 Signals are transduced by movement of macromolecules in the plane of the plasma membrane 5.4 Impact of the findings on cell signaling and molecular medicine 5.4.1 A novel paradigm of mammalian cell signaling 162 165 165 5.4.2 The physiological effect of MARCKS is mediated by the integral membrane pool of PKCα 166 5.4.3 Regulation of the oncogene Raf-1 by the integral membrane pool of PKCα 168 5.4.4 The effect of integral membrane PRK1 on progression of androgen dependent and independent prostate cancer 169 5.4.5 Integral membrane pool of PRK1 and PKCα as possible therapeutic targets for neurodegenerative diseases Bibliography 170 174 xiii List of Figures and Tables Figure 1.1 Major members of the serine/threonine kinase family and tyrosine family Figure 1.2 Dendrogram based on a sequence comparison of the PKC Superfamily Figure 1.3 Domain structures of the PKC subfamilies 10 Figure 1.4 Domain structure of PRK1 22 Figure 1.5 Proposed signaling pathways for GPCR activation of Rho 25 Figure 1.6 The structure of phospholipids 31 Figure 1.7 The membrane targeting of PKCα via C1 and C2 domains 41 Figure 3.1 Identification of an integral membrane pool of exogenous PRK1 in COS1 cells 75 Figure 3.2 Identification of an integral membrane pool of exogenous PRK1 by Triton X-114 phase partitioning 76 Figure 3.3 Identification of an integral membrane pool of endogenous PRK1 in cell lines 78 Figure 3.4 Endogenous PRK1 in rat erythrocytes is an integral membrane protein Figure 3.5 PRK1 is in lipid rafts of rat erythrocytes 79 80 Figure 3.6 Immunoelectron microscopy showing ultrastructural localization of PRK1 in rat liver 81 Figure 3.7 PRK1 is in the plasma membrane 82 Figure 3.8 PRK1 may have transmembrane domains 84 Figure 3.9 Topology of PRK1 in the plasma membrane 85 xiv Figure 3.10 Further characterization of membrane integration of PRK1 86 Figure 3.11 The integration of PRK1 with cellular membrane is independent of internal myristoylation and palmitoylation 87 Figure 3.12 The PRK1 mutant (S377A) is no longer an integral membrane protein Figure 3.13 Characterization of anti-P-P377 antibody 88 89 Figure 3.14 Myc-PRK1 with phosphoserine 377 is abundantly expressed in cells 90 Figure 3.15 Serine 377 is not an intramolecular autophosphorylation site 91 Figure 3.16 Characterization of PRK1-S377A mutant 93 Figure 3.17 Integral membrane PRK1 is catalytically competent 95 Figure 3.18 The dynamics of PRK1 at the plasma membrane of hepatocytes during liver regeneration 96 Figure 3.19 RhoA increases the activity of WT-PRK1 but not PRK1-S377A in COS1 cells 97 Figure 3.20 RhoA cannot increase the activity of the aqueous phase of PRK1 98 Figure 3.21 RhoA can increase the activity of PRK1 in the detergent-rich phase Figure 3.22 Activation of membrane-bound PRK1 by LPA 99 100 Figure 3.23 Wild-type and constitutively active RhoA are integral membrane proteins 102 Figure 3.24 Neurite retraction induced by RhoA is through the integral membrane pool of PRK1 103-104 Figure 3.25 Only the integral membrane pool of PRK1 can initiate transcriptional activation of the androgen receptor mediated by RhoA Figure 3.26 PRK1-S377A mutant is abundantly expressed in the cytoplasm Table 3.1 Reported increase in PRK1/PKN activity induced by RhoA 106 107 xv activation 109 Figure 4.1 Identification of an integral membrane pool of PKCα, PKCε and PKCζ 119 Figure 4.2 Catalytically inactive PKCα can integrate into membranes 120 Figure 4.3 Topology of PKCα in the plasma membrane 121 Figure 4.4 Mechanistic study of PKCα, PKCε and PKCζ integration into cellular membranes 122 Figure 4.5 V3 domain and C-terminal portion of C2 domain of PKCα are involved in membrane integration of PKCα 124 Figure 4.6 The integral membrane pool of ectopically expressed PKCα in COS1 cells is subjected to the acute regulation by TPA 125 Figure 4.7 The integral membrane pool of endogenous PKCα in NIH3T3 cells is subjected to acute regulation by TPA 126 Figure 4.8 The integral membrane pool of endogenous PKCα in NIH3T3 cells is subjected to acute regulation by DOG and ionomycin 127 Figure 4.9 The catalytic activities of various pools of ectopically expressed PKCα under basal conditions 128 Figure 4.10 The catalytic activities of various pools of endogenous PKCα in NIH3T3 cells under basal conditions 129 Figure 4.11 TPA cannot increase the catalytic activity of PKCα in the soluble fraction 130 Figure 4.12 The catalytic activity towards an S6 peptide of various pools of ectopically expressed PKCα in COS1 cells 131 Figure 4.13 The catalytic activities of various pools of endogenous PKCα and PKCε 132 Figure 4.14 Quantifications of dynamics of the peripheral membrane pool of PKC mutants 134 xvi Figure 4.15 Basal catalytic activity of PKCα mutants as determined in an in vitro kinase assay 135 Figure 4.16 PKCα-D294G and E265G mutants have no discernible defect in their catalytic activities 136 Figure 4.17 WT-PKCα and PKCα-D294G/E265G had indistinguishable distribution in cells Figure 4.18 Dynamics of GFP-MARCKS in CHO-K1 cells 138 140 Figure 4.19 PKCα-D294G failed to initiate cytoplasmic translocation and phosphorylation of MARCKS upon treatment with 100 nM TPA for 90 seconds 142 Figure 4.20 Only WT-PKCα but not PKCα-D294G was able to activate p42-MAPK in vivo 143 Figure 4.21 PKCα-D294G and PKCα-E265G failed to augment neurite outgrowth in N1E-115 neuronal cells 145 xvii Publications PAPERS IN REFEREED JOURNALS Yimin Zhu, Donna B. Stolz, Fengli Guo, Mark A. Ross, Simon C. Watkins, Bee Jen Tan, Robert Z. Qi, Ed Manser, Qiu Tian Li, Boon Huat Bay, Tian Seng Teo and Wei Duan, “Signaling via a novel integral plasma membrane pool of a serine/threonine protein kinase PRK1 in mammalian cells”, The FASEB Journal, 18:1722-1724, 2004. Yimin Zhu, Wee Guan Lim, Bee Jen Tan, Tian Seng Teo & Wei Duan, “Identification of an integral plasma membrane pool of protein kinase C in mammalian tissues and cells”, Cellular Signalling, available on line on January 15, 2005. Wee Guan Lim, Yimin Zhu, Chern-Hoe Wang, Bee Jen Tan, Jeffrey Armstrong, Terje Dokland , Hongyuan Yang, Yi-Zhun Zhu, Tian Seng Teo and Wei Duan, “The last five amino acid residues at the C-terminus of PRK1/PKN is essential for full lipid responsiveness”, Cellular Signalling, available on line on January 29, 2005 (as Co-first author). Yimin Zhu, Qihan Dong, Bee Jen Tan, Wee Guan Lim, Shufeng Zhou, Wei Duan., “ The PKCα-D294G mutant found in pituitory and thyroid tumors fails to transduce extracellular signals” , Cancer Research, 65 (11): 4520-4524, 2005 xviii W. Fan, J. Ding, W. Duan and Y. M. Zhu, “The influence of magnetic fields exposure on neurite outgrowth in PC 12 rat pheochromocytoma cells”, Journal of Magnetism and Magnetic Materials, 282 (2004) 325-328 CONFERENCE ABSTRACTS Yimin Zhu and Wei Duan “Rewriting the rule book of cell signalling---PRK1 serine/Threonine kinase is an integral membrane protein”, Life Sciences for Singapore— Third Combined Annual Scientific Meeting”, NUS, Singapore, p18, 2002. Yimin Zhu and Wei Duan “PRK1 is an integral membrane protein”, ASIA-PACIFIC CONFERENCE ON ANTI-AGEING MEDICINE 2002 June 23-26, 2002. Wei Duan and Yimin Zhu, “Signaling by integral membrane ser/thr kinases”, Signaling The Future, Biochemical Society, Liverpool, United Kingdom, p. 129, 2002. Yimin Zhu and Wei Duan, “Regulation of subcellular localization of PRK1 by Ser-377 phosphorylation”, Signaling The Future, Biochemical Society, Liverpool, United Kingdom, p. 74, 2002. Yimin Zhu and Wei Duan “A novel integral membrane pool of PRK1”, Gordon Research Conference-Molecular Biology. Tilton, USA, June 2003. W. Duan and Y. Zhu, “Exploring the potential role of PRK1/PKN in pathophysiology of Parkinson’s disease”, Movement Disorders 19, Suppl. 9: S28, 2004. (Impact Factor 2.895). xix Summary Members of the protein kinases C (PKC) superfamily, including PKCα and protein kinase Crelated kinase (PRK1), are known to play pivotal roles in many biological processes including proliferation, differentiation and apoptosis. In this study, a novel integral membrane pool of PKC superfamily of serine/threonine kinases has been identified by four procedures that use distinct underlying biochemical principles to remove loosely-bound membrane proteins from biological membranes. The integration of PRK1 into cellular membranes is found to be dependent, at least partially, on the phosphorylation status of the serine-377 residue. As for PKCα, the integrity of C2-V3 domain of this kinase is found to be critical for the integration of the protein into cellular membranes. The functional importance of the novel integral membrane pool of PRK1 and PKCα in mammalian cell signaling has been explored. Only the wild-type PRK1, but not the PRK1-S377A mutant that is no longer an integral membrane protein, is able to initiate RhoA-mediated and ligand-dependent transcriptional activation of the androgen receptor in human epithelial cells and to mediate RhoA-induced neurite retraction in mouse neuronal cells. Similarly, only the wild-type, but not the membrane-integration deficient PKCα mutants, is able to mediate phorbol esterstimulated translocation of MARCKS, to activate mitogen-activated protein kinase (MAPK) and to augment melatonin-stimulated neurite outgrowth. The findings presented in this thesis suggest that integral plasma membrane pools of intracellular serine/threonine protein kinases may play important roles in mediating cellular responses to extracellular stimuli. Selective modulation of the activity of the integral plasma xx membrane pool of serine/threonine kinases could constitute a novel strategy for clinical interventions of devastating diseases such as cancer, diabetes and autoimmune diseases. Abbreviations ADAM a disintegrin and metalloprotease AGC family protein kinase A, protein kinase G and protein kinase C AKAP A-kinase anchoring proteins aPKC atypical protein kinase C AR androgen receptor ATP adenosine 5’-triphosphate βARK β-adrenergic receptor kinase CaM calmodulin CaMK Ca2+/Calmodulin-dependent protein kinase cAMP adenosine 3’, 5’-cyclic monophosphate cGMP guanosine 3’, 5’-cyclic monophosphate CL cardiolipin CLK Cdk-like kinase cPKC conventional/classical protein kinase C CO carbon monoxide DAG diacylglycerol DOG sn-1, 2-dioctanoylglycerol EDTA ethylenediaminetetraacetic acid EGF epidermal growth factor ER endoplasmic reticulum xxi ERK extracellular signal-regulated kinase ERM ezrin, radixin, and moesin FGF fibroblast growth factor FLC furin-like convertase FPLC fast performance liquid chromatography FRET fluorescence resonance energy transfer GAP GTPase activating protein GDI guanine nucleotide dissociation inhibitor GDP guanosine 5’-diphosphate GEF guanine nucleotide exchange factor GFP green fluorescent protein GLUT4 glucose transporter GPCR G-protein coupled receptor GSK3 glycogen synthase kinase GST glutathione S-transferase GTP guanosine 5’-triphosphate HPLC high performance liquid chromatography HR homology region IP immunoprecipitation IP3 inositol-1,4,5-triphosphate IPTG isopropylthio-β-D-galactoside LORR loss of righting reflex LPA lysophosphatidic acid lysoPC lysophosphatidylcholine MARCKS myristoylated alanine-rich C-kinase substrate MAPK mitogen-activated protein kinase MBP myelin basic protein xxii MKK MAPK kinase NO nitric oxide nPKC novel protein kinase C PA phosphatidic acid PAK protease-activated kinases PC phosphatidylcholine PDGF platelet-derived growth factor PDK1 phosphoinositide-dependent kinase PE phosphatidylethanolamine PG phosphatidylglycerol PI phosphatidylinositol PI-3K phosphoinositide 3-kinase or phosphatidylinositol-3-kinase PICK-1 proteins that interact with C kinase-1 PIP2 phosphatidylinositol-4,5-bisphosphate PIP3 phosphatidylinositol-3,4,5-trisphosphate PKA cAMP-dependent kinase PKC protein kinase C PKG cGMP-dependent kinase PKN protein kinase N PLA phospholipase A PLC phospholipase C PLD phospholipase D PM plasma membrane PMA phorbol 12-myristate 13-acetate PMSF phenylmethylsulfonyl fluoride PRK protein kinase C-related kinase PS phosphatidylserine xxiii PSA prostate-specific antigen RACKs receptors for activated C kinase RICKs receptors for inactive C-kinases RKIP Raf-1 kinase inhibitor protein RP-HPLC reversed-phase high performance liquid chromatography RTK receptor tyrosine kinase S1P sphingosine-1-phosphate SBP substrate-binding protein SDS sodium dodeyl sulfate STICKs substrates that interact with C-kinases TM transmembrane TPA 12-O-tetradecanoylphorbol 13-acetate WT wild type [...]... alter nuclear and chromatin structure to induce apoptosis240 PKCα can also influence cell differentiation The inhibition of cell cycle progression and expression of cell- specific functions are the characteristics of cell differentiation PKCα is known to upregulate some proteins which affect cyclin and cyclin-dependent kinase to arrest the cell cycle to facilitate differentiation in MCF-10 human mammary... phosphorylated by PKCα and phosphorylation of Ser-153 relieves inhibition of the Raf/MAP kinase signaling cascade 234 Rho protein is also a downstream target of PKCα in the formation of lamellipodia 235 PKCα is involved in a variety of biological responses PKCα can regulate the proliferation of cells due to activation of the extracellular signal-regulated kinase/ mitogen-activated protein kinase (ERK/MAPK) cascade... bind to the region containing EF-handlike motifs of non-skeletal muscle type α-actinin in a Ca2+-sensitive manner, and to that of skeletal muscle type α-actinin in a Ca2+-insensitive manner PI-4,5P2 regulates the F-actingelating activity of α-actinin in vitro, and also activates the protein kinase activity of PRK1 in vitro228 PRK1 may be involved in the regulation of vesicle transport PRK1 was discovered... Phe) that are exposed by conformational changes of proteins283 and interactions between proteins and a lipid messenger such as DAG284 Specific domains in proteins have been identified to be involved in the interaction with cellular membranes, such as the C1 domain, C2 domain, PH domain and FYVE domain 35 1.4.3.2 Integral membrane proteins An integral membrane protein is a membrane protein that requires... epithelial cells238 Furthermore, PKCα is involved in cell migration and adhesion For example, PKCα is found to translocate and accumulate in the focal contacts241; PKCα can also phosphorylate ERM proteins (ezrin, radixin, and moesin) to mediate cell migration242 PKCα is important in carcinogenesis Overexpression of PKCα in human breast cancer cells results in a more aggressive and metastatic phenotype... the active site Activation of protein kinases occurs by dissociation of the regulatory domain from the catalytic domain For PRKs, which lack the C1 domain, the HR 1a motif is proposed to act as a pseudosubstrate site to inhibit kinase activity62 The C2 domain is found in the cPKCs immediately C- terminal to the C1 domain C2 domains are present in many other proteins, including the synaptotagmins, rabphilin- 3A, ... nucleus, some of which can translocate to this organelle depending on the cell type and activators166 Recombinant PKCα mutants that are devoid of the regulatory domain or C- terminal parts of the catalytic domain have been shown to 20 translocate to the nucleus of cells167 It was suggested that a nuclear targeting sequence of PKCα is present in the hinge and N-terminus of the catalytic domain The nuclear... DAG DAG can increase the affinity of PKC for PS 93 Similarly, Ca2+ increases the affinity of PKC for PS 93 The activation response curve of PKC by PS is sigmoidal PS concentrations of 15 mol % of total lipid in the plasma membrane are sufficient to totally activate PKC in the presence of saturating amounts of Ca2+ and DAG PKC interacts with multiple phosphatidylserine molecules The actual number of. .. have similar as well as distinct structural features that dictate their subcellular localization and functions in mammalian cells 1.3.3 Biochemical properties of PKC Certain features of the PKC isozymes can be deduced from the protein structures derived from the sequence of the cloned PKC cDNAs However, the greater body of information has been accumulated by detailed biochemical analyses of naturally... level of plasma membrane- associated enzyme73 A rise in either internal Ca2+ or DAG can cause cytosol -membrane translocation of PKC74,75 These results established a temporal correlation between the intracellular distribution of PKC and its putative activation state Phorbol esters are potent activators of PKCs They can mimic the effects of the natural activator of PKCs, diacylglycerol76 It is widely recognized . abundant in cells. Enzyme-linked receptors are single-pass transmembrane proteins with an extracellular ligand-binding domain and an intracellular catalytic or enzyme-binding domain. The great. cell. According to their subcellular localization, receptors are divided into two groups: cell surface receptors and intracellular receptors. Often, the signaling cell and target cell are of. a combination of small and large intracellular signaling molecules. The former are second messengers; the latter are called intracellular signaling proteins including G proteins, small GTPase