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Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute,1 Diabetes Unit, Lab of Clinical Investigation, National Center for Complementary and Alternative Medicine,2 Lab of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland 208923
Received 4 September 2003/ Returned for modification 8 October 2003/ Accepted 23 October 2003
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ABSTRACT |
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-dependent pathway. These data demonstrate that phosphorylation of serines 261 and 289, outside the GTP-binding region of Gem, controls its inhibition of Rho kinase ß and associated changes in the cytoskeleton. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunits, and Ras superfamily members (4), has been modified to DXWE in RGK proteins. The G3 domain is thought to participate in the binding and hydrolysis of GTP as a result of the invariant aspartate-binding catalytic Mg2+ and the amide proton of the invariant glycine forming a hydrogen bond with the 
-phosphate of GTP (21). Consistent with substitution of the glycine residue, the intrinsic GTPase activity of recombinant Gem (7) or Rem (11) appears to be at the border of detection and considerably less than that of Ras.
Another consideration is the inherent flexibility of the Gly residue in the DXXG motif, which appears to be important for allowing a conformational change in the G3 loop and downstream helix following GTP binding (summarized in reference 4). In Gs, replacement of Gly with Ala in the G3 region prevents the activation of downstream effectors without preventing binding of GTP (19). Therefore, it is an open question as to whether RGK family members undergo conformational shifts between GDP and GTP bound states similar to other GTPases.
Other mechanisms of regulation related to unique structural features of the RGK proteins likely exist. RGK family members contain carboxyl extensions of approximately 40 amino acids. These extensions contain a calmodulin binding site (12, 20) and a perfectly conserved terminal sequence of 10 amino acids. Phosphorylations of the carboxyl extensions of Gem (17, 20), Rad (20), and Rem (9) have been described. In addition, the COOH terminus of Rem has been reported to bind 14-3-3 (9), although the specific binding site was not determined. Amino-terminal extensions are more variable in length and sequence among the family members. Finally, unlike most Ras superfamily members, at least some RGK proteins appear to be regulated at the level of protein expression. Gem is an early response gene that is dramatically regulated at the transcriptional level (17), and Rad protein levels are regulated by posttranslational processes (32).
Recent advances in defining functional assays for Gem have made it possible to begin addressing the regulation of this RGK protein. The development of cell-based assays has followed from the separate descriptions of two Gem-binding proteins, the voltage-gated calcium channel ß subunits (3) and Rho kinase ß (30). Both proteins bind Gem within the core region. Gem down-regulates voltage-gated calcium channel activity, which exists mainly in neuronal and endocrine cells (3). One mechanism that has been suggested is that Gem binds and sequesters the ß subunit, resulting in inhibition of -subunit expression at the plasma membrane (3).
Gem is also expressed in cell types that do not express voltage-gated calcium channels, and a role for Gem in regulating cytoskeletal alterations has been observed in various cell types (1, 16, 23). In addition to ROKß, Gem has been shown to bind KIF-9, a kinesin-like protein (23), and Gmip, a RhoGAP-containing protein (1). Whether KIF-9 or Gmip affects cytoskeletal morphology and the role of Gem in regulating these proteins is yet to be established. Gem regulation of cytoskeletal alterations mediated by ROKß has been demonstrated. Ectopic Gem expression inhibits ROK-mediated formation of stress fibers, focal adhesions, and neurite retraction (30). Consistent with inhibiting endogenous ROKß, Gem stimulates neurite extension and cell flattening in neuroblastoma cells (16). Gem inhibits the ability of ROKß to phosphorylate myosin light chain and myosin phosphatase, but not LIM kinase, suggesting that the ROKß-Gem interaction blocks the interaction of ROK with specific substrates (30). Because cytoskeletal function is involved in the intracellular transport of surface-bound receptors (27), one question has been whether calcium channel ß subunits and ROK represent entirely separate effector pathways or whether they might exist together in a supramolecular complex.
Here we show that serine-to-alanine point mutations in serines 261 and 289 within the COOH terminus of Gem, outside the Ras-related GTP-binding core and ROKß-binding domain, lead to a complete loss of Gem-mediated cytoskeletal reorganization. By contrast, inhibition of voltage-gated calcium channel activity is unaffected by these same mutations but instead is reversed in GTP- and calmodulin-binding mutants. Thus, pathways affecting calcium channels and Rho kinase appear to be separable. Data presented suggest that COOH-terminal phosphorylation, in addition to regulating cytoskeletal function, results in a conformational change that is stabilized by bidentate 14-3-3 binding, leading to an increased half-life for the phosphorylated Gem protein.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inhibitors. The kinase inhibitors Go6976, Go6850, H9 dihydrochloride, KT5720, PD98059, and KN93 were purchased from Calbiochem (San Diego, Calif.). W7 was from Biomol Research Laboratories, Inc. (Plymouth Meeting, Pa.), LY294002 was from Biosource International, Inc. (Camarillo, Calif.), and Y27632 was from Welfide Corp. (Tokyo, Japan). KT5823 and Apigenin were provided by Alexis Biochemicals (San Diego, Calif.).
Plasmids.
A QuickChange site-directed mutagenesis kit (Stratagene, LaJolla, Calif.) was used to generate the mutants of Gem in pMT2T and pRC-CMV vectors. Mutagenized inserts were verified by sequence determination. A dominant inhibitory phosphatidylinositol 3-kinase (PI 3-kinase) regulatory subunit, p85(
479-513), was described previously (24). Constitutively active protein kinase C (PKC) was generated by mutating Ala 119 to Asp in the pseudosubstrate region (28). PKC kinase dead is a mutation of Leu 281 to Trp (25). Wild-type phosphoinositide-dependent kinase-1 (PDK-1) and kinase-inactive PDK-1(K114A) have been described elsewhere (5).
Transfections, immunoprecipitations, and Western blot analysis. Cos-7 cells were plated at 2.0 x 106 cells/10-cm cell culture plate in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were transfected with 4 µg of total DNA using Lipofectamine Plus following the Invitrogen protocol. Soluble protein extracts were prepared as previously described (16) with the addition of 4 nM calyculin, 25 mM ß-glycerophosphate, and 10 mM NaF. The extract was precleared on 50 µl of recombinant protein G-agarose beads (Invitrogen) for 30 min at 4°C, followed by an incubation of the precleared lysate with 25 µl of packed beads (per sample) and 12 µg of anti-Gem monoclonal antibody P2D10 for 2 to 4 h at 4°C. Beads were then washed three times in lysis buffer, resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and boiled for 2 min. SDS-PAGE gels were run and electroblotted onto polyvinylidene difluoride membranes, and Western blot analysis was performed.
Calmodulin binding. To perform the calmodulin-binding experiments, cells were transfected, harvested 48 h later, and lysed as described above. The cellular extract was precleared on glutathione-Sepharose 4B beads (Amersham Bioscience, Piscataway, N.J.) for 30 min at 4°C. The precleared extract was subsequently incubated with 25 µl of packed calmodulin-Sepharose 4B beads (Amersham Bioscience) for 4 h at 4°C. The beads were then washed three times with lysis buffer, resuspended in SDS-PAGE sample buffer, and boiled for 2 min. Western blot analysis was performed with anti-Gem polyclonal antibodies.
Electrophysiology of dissociated SCG neurons.
Single superior cervical ganglion (SCG) neurons were isolated from 10- to 14-week-old male Wistar rats as previously described (14). A mammalian expression vector pRC-CMV containing the wild-type or mutant GEM cDNA (0.1 µg/µl in 10 mM Tris [pH 8]) was injected into the nucleus of SCG neurons 4 to 
8 h following cell isolation with an Eppendorf FemtoJet microinjector and a 5171 micromanipulator (14). Neurons were coinjected with enhanced green fluorescent protein (EGFP) cDNA (5 ng/µl; pEGFP-N1; Clontech Laboratories, Palo Alto, Calif.) to facilitate later identification of successfully injected cells. Neurons were used for recording 20 to 24 h after injection.
Whole-cell currents were recorded with a patch-clamp amplifier (Axopatch 200B; Axon Instruments) at room temperature (21 to 24°C) using the conventional variant of the patch-clamp technique (13) as previously described (15). For recording calcium current, patch electrodes were pulled from borosilicate glass capillaries (Corning 7052; Garner Glass, Claremont, Calif.) on a P-97 Flaming-Brown micropipette puller (Sutter Instruments, Midland, San Rafael, Calif.), fire polished on a microforge, and filled with a solution containing 120 mM N-methyl-D-glutamine, 20 mM tetraethylammonium-OH, 10 mM HEPES, 11 mM EGTA, 1 mM CaCl2, 14 mM Tris-creatine phosphate, 4 mM Mg-ATP, and 0.3 mM Na2-GTP; pH was adjusted to 7.24 with methanesulfonic acid and HCl (final Cl- concentration of 20 mM). Pipette resistance ranged from 1 to 3 M
, yielding uncompensated series resistances of 2 to 6 M. Series resistance compensation of 80% was used in all recordings. The external solution consisted of 145 mM tetraethylammonium methanesulfonate, 10 mM HEPES, 15 mM glucose, 10 mM CaCl2, and 300 nM tetrodotoxin (pH 7.35 and 320 mosmol/kg). Voltage protocol generation and data acquisition were performed using custom data acquisition software on a Macintosh computer with an ITC-18 interface board (InstruTECH, Port Washington, N.Y.). Ica traces were low pass filtered at 2 to 5 kHz and digitized at 10 kHz. For IM recordings, pipettes were pulled from borosilicate glass capillaries (1B150F-4; World Precision Instruments, Sarasota, Fla.) and filled with a solution consisting of 140 mM KCl, 0.1 mM EGTA, 10 mM HEPES, 4 mM Mg-ATP, 0.1 mM Na2-GTP, 0.1 mM Tris-creatine phosphate (pH adjusted to 7.25 with KOH; 300 mosmol/kg). The external solution contained 150 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 1 mM MgCl2, 1 mM CaCl2, 15 mM glucose, 300 nM tetrodotoxin (pH adjusted to 7.35 with NaOH; 325 mosmol/kg). IM traces were low pass filtered at 1 kHz and digitized at 2 kHz. Drugs were applied by positioning a silica tube (inner diameter, 200 µm) of a custom-designed gravity-fed microperfusion system at 100 µm from the cell. Oxotremorine-M (OXO-M) and norepinephrine were purchased from Sigma (St. Louis, Mo.).
Ca2+ current traces and current-voltage relationships were corrected for linear leakage current as determined from hyperpolarizing pulses. Ica amplitude was determined 10 ms after the onset of a test pulse. M current traces were not leak subtracted. Numerical values are expressed as the mean ± the standard error of the mean (SEM). Statistical comparisons were made by using Student's t test or analysis of variance followed by Dunnett's test. The differences were considered significant if P was 
0.05.
T-cell activation. Human T cells were plated (3.0 x 106 cells/10-cm cell culture plate) in RPMI 1640 medium with 10% fetal bovine serum. Cells were stimulated with the addition of 20 ng of phorbol myristate acetate/ml and 5 µg of phytohemagglutinin (Sigma)/ml and incubated for various times. Three plates of each time point were combined, washed with phosphate-buffered saline (PBS), and analyzed for Gem protein using the immunoprecipitation and Western blotting protocols described above.
Immunofluorescence. Exponentially growing HeLa cells were plated on glass coverslips (A. Daigger & Co.) in 24-well cell culture dishes and incubated overnight. The following day, cells on each coverslip were transfected with 0.5 µg of PMT2T-Gem, using Lipofectamine Plus (Invitrogen Life Technologies). Cells were then fixed with 4% paraformaldehyde for 10 min at room temperature, rinsed three times with PBS, and permeabilized with 1% Triton X-100 in 0.2% bovine serum albumin (BSA)-PBS for 2 min at room temperature. Cells were stained using polyclonal anti-Gem antibody followed by antivinculin monoclonal antibody staining as described previously (30). Stained cells were examined on a Zeiss Axioplan microscope equipped with a 100x/1.4 oil immersion objective, and confocal images were generated on a Zeiss LSM 510.
Neuroblastoma neurite extension assay. Neurite remodeling resulting from transfection of N1E-115 murine neuroblastoma cells with Gem or Gem mutants was assayed as described previously (16). Transfections were done using Lipofectamine Plus.
PC12 cell culture and transfection. PC12 cells were maintained in Dulbecco's modified Eagle medium supplemented with 7.5% heat-inactivated fetal bovine serum and 7.5% heat-inactivated horse serum at 37°C and 5% CO2. Cells were plated on collagen IV-coated six-well cell culture dishes (BD Biosciences) at a concentration of 106 cells per well. Twenty-four hours later, cells were transiently transfected with 8 µg of PMT2T-based expression plasmid and 2 µg of PCDNA3-hGH using Lipofectamine 2000 (Invitrogen Life Technologies). Twenty-four hours following transfection, PC12 cells were fed with fresh culture medium and incubated overnight at 37°C and 5% CO2. Forty-eight hours following transfection, PC12 cells were rinsed twice with physiological salt solution (PSS) containing 20 mM HEPES (pH 7.4), 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 1 mM KH2PO4, 10 mM glucose, and 0.1% BSA. Cells were preincubated in PSS for 30 min. Nonstimulated secretion was investigated by incubating PC12 cells in PSS for another 30 min, while depolarization-induced secretion was shown by incubating cells for 30 min in a high-KCl solution containing 20 mM HEPES (pH 7.4), 65 mM NaCl, 80 mM KCl, 2.5 mM BaCl2, 1 mM MgCl2, 1 mM KH2PO4, 10 mM glucose, and 0.1% BSA. Growth hormone protein concentration was determined by Western blot analysis of cell-conditioned medium using polyclonal anti-human growth hormone (hGH) antibody obtained from A. F. Parlow (NIDDK National Hormone and Peptide Program). To determine the cellular hGH, at the end of the incubations cells were lysed in 25 mM HEPES (pH 7.4), 10% glycerol, 1% Triton X-100, 150 mM NaCl.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In order to address whether Gem binds specific isoforms of 14-3-3, we determined the coassociation of Gem and 14-3-3 in the neuroblastoma cell line SY5Y. Western blotting was performed on Gem immunoprecipitates using isoform-specific antibodies to 14-3-3. 14-3-3, -, -
, and -ß were observed to bind to Gem (data not shown). Therefore, no specificity was apparent for the binding of these 14-3-3 isoforms to Gem.
Phosphorylation of serines 261 and 289 is required for Gem-mediated neurite extension in N1E-115 cells. Our investigators have shown that Gem stimulates cell flattening and neurite extension in NIE-115 cells (reference 30 and Fig. 2A) and opposes Rho kinase ß-mediated cell rounding, a function that is dependent upon the interaction of Gem and ROKß. In order to determine whether phosphorylation of serines 261, 287, 289, or 294 in the COOH-terminus affects such function, we tested the appropriate serine-to-alanine Gem mutations for their ability to stimulate flattening and neurite extension in transfected N1E-115 cells (Fig. 2B) and opposition to ROKß-stimulated neurite retraction (data not shown). Interestingly, mutation of either serine 289 or serine 261 to alanine inhibited the morphological activity of Gem, whereas mutation of serines 287 and 294 had no effect (Fig. 2B). In order to mimic the structure of phosphoserine, serines 261 and 289 were mutated to aspartic acids. Both of these mutants stimulated neurite extension (Fig. 2B) and opposed ROKß (data not shown) similarly to wild type. To determine whether mimicking constitutive phosphorylation with aspartic acid at one site (serine 261 or 289) could overcome the lack of phosphorylation at the other site, the double mutants Gem261D289A and Gem261A289D were assayed in the N1E-115 system. Neither mutant showed activity, suggesting that both sites need to be phosphorylated or modified with a negative charge to stimulate neurite extension.
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Neither phosphorylation of serine 261 nor of 289 influences calmodulin binding. Gem binds calmodulin in the COOH terminus, between serines 261 and 289. To test whether serine phosphorylation and/or 14-3-3 binding affects calmodulin binding, the ability of various Gem mutants to bind calmodulin-Sepharose was determined. A mutation from tryptophan to glycine at position 269 in mouse Gem (or the equivalent position 270 in human Gem) within the helix that binds calmodulin inhibits its binding. Here, the calmodulin-binding mutant is labeled GemW269G to remain consistent with the published literature (3, 12). As shown in Fig. 3, with the exception of GemW269G, the various mutants bound calmodulin-Sepharose. Therefore, phosphorylation at either site 261 or 289 within the COOH terminus is not required for nearby calmodulin binding.
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To further analyze the physiological role of Gem, we examined the effect of wild-type Gem and various Gem mutants in PC12 cells in which Ca2+ influx through L-type calcium channels triggers GH secretion. As shown in Fig. 5, the effect of Gem on Ca2+-stimulated secretion mostly paralleled the results observed in direct measurements of channel activity. Wild-type Gem inhibited secretion. GemS89N, which has reduced affinity for GTP (data not shown), failed to inhibit secretion. GemW269G partially inhibited secretion, and GemS261A inhibited secretion to the same extent as wild type. It was not possible to evaluate GemS289A in PC12 cells, because the Gem mutant protein was particularly unstable in this cell type (Fig. 5, Gem expression, bottom panel). Together these data show that the cytoskeletal reorganization function of Gem does not play a role in Ca2+-triggered secretion, despite the obvious role of the cytoskeleton in exocytosis.
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To determine the phosphorylation status of serines 261 and 289 in endogenous Gem, we assayed reactivity of the anti-pS-261 and -pS-289 peptide antibodies toward Gem induced by T-cell receptor activation in normal peripheral blood T cells. As shown in Fig. 8, Gem was induced and accumulated with time after mitogenic activation of T cells. Phosphorylation of serines 261 and 289 could be observed at the earliest time point of Gem protein expression. Serine 261 phosphorylation appeared to reach peak expression with slightly delayed kinetics relative to serine 289.
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activation.
We used protein kinase inhibitors in combination with anti-pS-289 antibodies to screen for the kinases responsible for phosphorylation of serine 289 in N1E-115 or Cos cells transfected with GemS23A. GemS23A was used in order to reveal dynamic dephosphorylation of serine 289, which is probably inaccessible to phosphatases following 14-3-3 binding. The list of inhibitors used to examine potential kinases responsible for phosphorylation of serine 289 is shown in Table 1. None of the drugs employed appeared to inhibit serine 289 phosphorylation under the conditions that were used (data not shown). The context of serine 289, amino acid sequence SCHD, is a potential target site for casein kinase II, but apigenin, a casein kinase II inhibitor, did not diminish phosphorylation at serine 289. This suggests that a separate class of enzymes from those tested are responsible for phosphorylation of serine 289 or, more likely, that more than one kinase phosphorylates serine 289 in Cos cells.
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greatly increased pS-261 reactivity (Fig. 9B). A well-studied pathway leading to aPKC activation is the PI-3 kinase/PDK-1 pathway. However, phosphorylation of Ser 261 was not reduced following treatment with the PI 3-kinase inhibitor LY294002 but, surprisingly, appeared to increase (Fig. 9A). In addition, phosphorylation of Ser 261 was unaffected following cotransfection of either a mutant PI-3 kinase p85 subunit (p85479-513) that constitutively inhibits activation of the p110 catalytic subunit or a dominant-negative form of PDK-1 (Fig. 9B). Therefore, it appears that PKC-dependent phosphorylation of Gem 261 is not downstream of PI-3 kinase/PDK-1 in Cos cells.
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in complex with PAR6 (8). As shown in Fig. 9C, coexpression of constitutively activated cdc42 resulted in increased S261 phosphorylation of Gem, which was inhibited by Go6850 treatment, suggesting that cdc42-stimulated PKC activation leads to Gem 261 phosphorylation. Constitutively activated rac did not result in a similar increase in Gem S261 phosphorylation (data not shown). Consistent with a role for the cdc42 pathway in GemS261 phosphorylation, dominant-negative cdc42 partially inhibited phosphorylation of constitutively expressed Gem. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gem inhibition of high-voltage-activated (HVA) Ca2+ channel activity is most likely mediated through an interaction with the HVA Ca2+ auxiliary ß subunit isoforms. The finding that M-type K+ channels are unaffected by Gem expression is consistent with this notion and rules out a general nonspecific suppression of ion channel function. The in vitro interaction of Gem with the ß subunit previously has been shown to be dependent upon GTP loading of Gem (3), and the demonstration here that the GTP-binding mutant GemS89N has no functional activity is consistent with the notion that GTP binding regulates the ß subunit-Gem interaction. HVA Ca2+ channel inhibition appears similar to a classical small GTPase effector pathway in that GTP binding is required, although the GTPase activity of Gem is unlikely, based upon sequence comparisons to known GTPases. Defining a mechanism for regulating GDP-GTP exchange on Gem is an important question yet to be answered.
In addition, unique regulatory mechanisms such as calmodulin binding exist for Gem and other RGK family members (12, 20). Calmodulin binding in the C-terminal extension of Gem is required for maximal inhibition of HVA Ca2+ channels by ectopically expressed Gem, as determined by measurement of electrical activity in primary neurons and by Ca2+-evoked secretion in PC12 cells (Fig. 4 and 5) (3). The suggestion has been made that Gem inhibits HVA Ca2+ channel activity as a result of sequestering the ß subunit and thereby inhibiting transport of the subunit to the cell surface. However, the relatively fast kinetics of virtually complete inhibition within 20 h suggest that a direct action of Gem upon the cell surface-localized channel should be considered.
The second functional property of Gem, Rad, and Rem is their stimulation of cytoskeletal reorganization (16, 22, 23, 30). As shown here, the cytoskeletal function of Gem, mediated by an inhibition of ROKß, is regulated by phosphorylations at serines 289 and 261 in the C-terminal extension of Gem. Mutation of either serine 261 or 289 to alanine resulted in a loss of Gem function in stimulating neurite extension, inhibiting focal adhesion formation, or reversing ROKß-mediated neurite retraction (Fig. 2). Mutation of the same serines to aspartic acids, which potentially mimic phosphoserine structure, did not disrupt these cytoskeletal functions. We have found no evidence that phosphorylation of either serine 261 or 289 alters the subcellular distribution of Gem (data not shown). The interaction of Gem with ROKß has been mapped to the core region of Gem in yeast two-hybrid analyses (30). Phosphorylation of serines 261 and 289 most likely leads to a conformational change in Gem, revealing binding sites for ROKß or other interacting proteins mediating the ROKß-dependent function of Gem. The in vivo interaction of Gem and ROKß is not of sufficiently high affinity to definitively address this question with coprecipitation analyses.
14-3-3 binding to Gem occurs as a result of bidentate binding to phosphoserine 23 in the N-terminal extension and phosphoserine 289 in the C-terminal extension. Although phosphorylation of serine 289 is required for cytoskeletal reorganization, subsequent 14-3-3 binding is not required as evidenced by the retention of activity in GemS23A and GemS289D mutants. Perhaps a conformational change stimulated by serine 289 phosphorylation is sufficient for activity of overexpressed Gem. As shown in Fig. 7B, 14-3-3 binding partially protects phosphoserine 289 from dephosphorylation. In addition, 14-3-3 dimers are rigid structures which are believed to lock their binding partners into specific conformations (31). 14-3-3-bound Gem has a twofold-longer half-life than nonbound Gem (Fig. 6). A similar increase in protein stability following 14-3-3 binding has been described for the Wee1 kinase (29). We expect that the role of 14-3-3 in maintaining an active conformation for Gem and/or regulating the level of Gem protein could be quite significant under conditions where Gem is limiting. Gem protein levels are subject to rapid fluctuations as a result of transcriptional control of Gem mRNA production and a short protein half-life (17). Under such conditions, phosphorylation of serine 289 and 14-3-3 association may indirectly regulate HVA Ca2+ channels through modulating the cellular concentration of Gem.
The combined use of PKC inhibitors and phosphopeptide-specific antibodies has revealed a role for aPKCs in GemS261 phosphorylation. In addition, in Cos cells it appears that stimulation of PKC through cdc42 activation leads to GemS261 phosphorylation, but stimulation of PKC through PI-3 kinase activation does not. The availability of Gem for PKC-dependent phosphorylation most likely is dependent upon appropriate localization of both proteins via scaffolding complexes within the cell. The biochemical mechanism whereby cdc42 activates PKC in the context of PAR6 has not been established (8). cdc42 is important for orienting actin polymerization at the leading edge of migrating cells (18), and cdc42 in conjunction with PKC, PAR6, and PAR3 is important in determining cell polarity (8). Activation of aPKCs has been shown to result in depolymerization of actin stress fibers downstream of cdc42 activation (6). Thus, the regulation of Gem by PKC-dependent phosphorylation of S261, leading to Gem inhibition of ROKß function, is consistent with a regulatory circuit that segregates actin polymerization and stress fiber formation.
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FOOTNOTES |
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