Actin modulation of a MARCKS phosphorylation site located outside the effector domain

MARCKS (Myristoylated alanine-rich C kinase substrate) is a ubiquitous actin regulating protein, especially abundant in the nervous system. This protein may be phosphorylated by other enzymes, particularly by proline-directed kinases, at serine and threonine residues located at different sites along its chain. We demonstrate here that the phosphorylation of chick MARCKS at serine 25, which only takes place in the nervous tissue, does not impair its association with particular plasma membrane regions such as the “detergent resistant microdomains” that also contain actin. This phosphorylated form of MARCKS is able to bind actin, and the integrity of actin filaments in cells (retina neuroblasts) is a necessary condition to sustain this phosphorylation. Taken together, these results indicate the existence of a functional interaction between actin filaments and MARCKS in cells, and particularly of an action in maintaining a phosphorylation in a region of the N-terminal moiety of MARCKS.     

Thrombin-induced phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein in bovine pulmonary artery endothelial cells

Myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate that is targeted to the plasma membrane by an amino-terminal myristoyl group. In its nonphosphorylated form, MARCKS cross-links F-actin and binds calmodulin (CaM) reciprocally. However, upon phosphorylation by PKC, MARCKS releases the actin or CaM. MARCKS may therefore act as a CaM sink in resting cells and regulate CaM availability during cell activation. We have demonstrated previously that thrombin-induced myosin light chain (MLC) phosphorylation and increased monolayer permeability in bovine pulmonary artery endothelial cells (BPAEC) require both PKC- and CaM-dependent pathways. We therefore decided to investigate the phosphorylation of MARCKS in BPAEC to ascertain whether this occurs in a temporally relevant manner to participate in the thrombin-induced events. MARCKS is phosphorylated in response to thrombin with a time course similar to that seen with MLC. As expected, MARCKS is also phosphorylated by phorbol 12-myristate 13 acetate (PMA), a PKC activator, but with a slower onset and more prolonged duration. Bradykinin also enhances MARCKS phosphorylation in BPAEC, but histamine does not. MARCKS is distributed evenly between the membrane and cytosol in BPAEC, and neither thrombin nor PMA caused significant translocation of the protein. Specific PKC inhibitors attenuated MARCKS phosphorylation by either thrombin or PMA. Since thrombin-induced MLC phosphorylation is also attenuated by these inhibitors, MARCKS may be involved in MLC kinase activation and subsequent BPAEC contraction. W7, a CaM antagonist, enhances the phosphorylation of MARCKS. This was expected since CaM binding to MARCKS has been shown to decrease MARCKS phosphorylation by PKC. On the other hand, tyrosine kinase inhibitors, genistein and tyrphostin, attenuate MARCKS phosphorylation but have no effect on MLC phosphorylation, suggesting that MARCKS may be phosphorylated by kinases other than PKC. Phosphorylation of MARCKS outside the PKC phosphorylation domain would not be expected to induce the release of CaM. These data provide support for the hypothesis that MARCKS may serve as a regulator of CaM availability in BPAEC. © 1996 Wiley-Liss, Inc.     

Association of PP1 with its regulatory subunit AKAP149 is regulated by serine phosphorylation flanking the RVXF motif of AKAP149

Reformation of the nuclear envelope at the end of mitosis involves the recruitment of the B-type lamin phosphatase PP1 to nuclear membranes by A-kinase anchoring protein AKAP149. PP1 remains associated to AKAP149 throughout G1 but dissociates from AKAP149 when AKAP149 is phosphorylated at the G1/S transition. We examine here the role of phosphorylation of serines flanking the RVXF PP1-binding motif of AKAP149, on PP1 anchoring. The use of AKAP149 peptides encompassing the RVXF motif and five flanking serines, either wild type (wt) or bearing S-->A or S-->D mutations, specifically shows that phosphorylation of S151 or S159 abolishes PP1 binding to immobilized AKAP149. Peptides with S151 or S159 as the only wt serine residue trigger dissociation of PP1 from immunoprecipitated AKAP149, whereas S151/159D mutants are ineffective. Furthermore, immunoprecipitated AKAP149 from purified G1-phase nuclear envelopes binds PKA and PKC in overlay assays. PKA binding to AKAP149 in vitro is unaffected by the presence of PKC or PP1, and similarly, PKC binding is independent of PKA or PP1. The immunoprecipitated AKAP149 complex contains PKA and PKC activities. Both AKAP149-associated PKA and PKC serine-phosphorylate immunoprecipitated AKAP149 in vitro; however, only PKC-mediated phosphorylation promotes dissociation of PP1 from the AKAP. The results suggest a putative temporally and spatially controlled mechanism promoting release of PP1 from AKAP149. AKAP149 emerges as a scaffolding protein for multiple protein kinases and phosphatases that may be involved in the integration of intracellular signals that converge at the nuclear envelope.     

MARCKS dephosphorylation is involved in bradykinin-induced neurite outgrowth in neuroblastoma SH-SY5Y cells

Bradykinin (BK) plays a major role in producing peripheral sensitization in response to peripheral inflammation and in pain transmission in the central nerve system (CNS). Because BK activates protein kinase C (PKC) through phospholipase C (PLC)-β and myristoylated alanine-rich C kinase substrate (MARCKS) has been found to be a substrate of PKC, we explored the possibility that BK could induce MARCKS phosphorylation and regulate its function. BK stimulation induced transient MARCKS phosphorylation on Ser159 with a peak at 1 min in human neuroblastoma SH-SY5Y cells. By contrast, PKC activation by the phorbol ester phorbol 12,13-dibutyrate (PDBu) elicited MARCKS phosphorylation which lasted more than 10 min. Western blotting analyses and glutathione S-transferase (GST) pull-down analyses showed that the phosphorylation by BK was the result of activation of the PKC-dependent RhoA/Rho-associated coiled-coil kinase (ROCK) pathway. Protein phosphatase (PP) 2A inhibitors calyculin A and fostriecin inhibited the dephosphorylation of MARCKS after BK-induced phosphorylation. Moreover, immunoprecipitation analyses showed that PP2A interacts with MARCKS. These results indicated that PP2A is the dominant PP of MARCKS after BK stimulation. We established SH-SY5Y cell lines expressing wild-type MARCKS and unphosphorylatable MARCKS, and cell morphology changes after cell stimulation were studied. PDBu induced lamellipodia formation on the neuroblastoma cell line SH-SY5Y and the morphology was sustained, whereas BK induced neurite outgrowth of the cells via lamellipodia-like actin accumulation that depended on transient MARCKS phosphorylation. Thus these findings show a novel BK signal cascade-that is, BK promotes neurite outgrowth through transient MARCKS phosphorylation involving the PKC-dependent RhoA/ROCK pathway and PP2A in a neuroblastoma cell line.     

Structural characterization of a neuroblast-specific phosphorylated region of MARCKS

MARCKS (Myristoylated Alanine-Rich C Kinase substrate) is a natively unfolded protein that interacts with actin, Ca^2 +–Calmodulin, and some plasma membrane lipids. Such interactions occur at a highly conserved region that is specifically phosphorylated by PKC: the Effector Domain. There are two other conserved domains, MH1 (including a myristoylation site) and MH2, also located in the amino terminal region and whose structure and putative protein binding capabilities are currently unknown. MH2 sequence contains a serine that we described as being phosphorylated only in differentiating neurons (S25 in chick). Here, Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy were used to characterize the phosphorylated and unphosphorylated forms of a peptide with the MARCKS sequence surrounding S25. The peptide phosphorylated at this residue is recognized by monoclonal antibody 3C3 (mAb 3C3). CD and NMR data indicated that S25 phosphorylation does not cause extensive modifications in the peptide structure. However, the sharper lines, the absence of multiple spin systems and relaxation dispersion data observed for the phosphorylated peptide suggested a more ordered structure. Surface Plasmon Resonance was employed to compare the binding properties of mAb 3C3 to MARCKS protein and peptide. SPR showed that mAb 3C3 binds to the whole protein and the peptide with a similar affinity, albeit different kinetics. The slightly ordered structure of the phosphorylated peptide might be at the origin of its ability to interact with mAb 3C3 antibody, but this binding did not noticeably modify the peptide structure.     

Phosphorylation of the MARCKS Protein (P87), a Major Protein Kinase C Substrate, Is Not an Obligatory Step in the Mitogenic Signaling Pathway of Basic Fibroblast Growth Factor in Rat Oligodendrocytes

Basic fibroblast growth factor (bFGF) is a well-characterized peptide hormone that has mitogenic activity for various cell types and elicits a characteristic set of responses on the cell types investigated. In this report we confirmed that bFGF is a potent mitogen for rat brain-derived oligodendrocyte (OL) precursor cells as well as for differentiated OL in secondary culture. bFGF was shown to induce expression of the protooncogene c-fos in OL. The role of protein kinase C (PKC) in mediating bFGF-stimulated proliferation as well as c-fos expression in OL was investigated. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated c-fos expression but did not trigger cell proliferation. When PKC was down-regulated by pretreatment of OL with PMA for 20 h, the bFGF-mediated stimulations of OL proliferation and c-fos mRNA expression were still observed, whereas the induction of c-fos mRNA by PMA was totally inhibited. These data demonstrate that the bFGF mitogenic signaling pathway in OLs does not require PKC. On the other hand, bFGF was found to stimulate specifically the phosphorylation of a limited number of PKC substrates in oligodendroglial cells, including the MARCKS protein. The bFGF-dependent phosphorylation of MARCKS protein was totally inhibited when PKC was first down-regulated, indicating that the phosphorylation of this protein is PKC dependent. Tryptic digestion of the phosphorylated MARCKS protein revealed that bFGF stimulated specifically the phosphorylation of the MARCKS protein on a single phosphopeptide. We provide evidence that bFGF also stimulated fatty acylation of the MARCKS protein, which might explain the observed specific bFGF-dependent phosphorylation of this protein in OL. We propose that bFGF-dependent fatty acylation and phosphorylation of the MARCKS protein are not essential for the transduction of the bFGF mitogenic signal but are probably linked to differentiation processes elicited by bFGF on OL.     

Inhibition of CD3-linked phospholipase C by phorbol ester and by cAMP is associated with decreased phosphotyrosine and increased phosphoserine contents of PLC-gamma 1.

The mechanisms by which phorbol 12-myristate 13-acetate (PMA) and cAMP attenuate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) induced by ligation of the T-cell antigen receptor complex (TCR) was studied in the human Jurkat T-cell line. It has previously been shown that stimulation of Jurkat cells with antibodies to CD3, components of the TCR, elicits a rapid and transient phosphorylation of phospholipase C (PLC)-gamma 1, the predominant PLC isozyme in Jurkat cells, at multiple tyrosine residues and that such tyrosine phosphorylation leads to activation of PLC-gamma 1. Prior incubation of Jurkat cells with PMA or forskolin, which increases intracellular cAMP concentrations, prevented tyrosine phosphorylation of PLC-gamma 1 as well as the hydrolysis of PtdIns 4,5-P2 induced by ligation of CD3. Dose-response curves of PMA and of forskolin for the inhibition of PLC-gamma 1 tyrosine phosphorylation and of PtdIns 4,5-P2 hydrolysis were similar. These results suggest that the inhibition of PtdIns 4,5-P2 hydrolysis by PMA and cAMP is attributable to reduced tyrosine phosphorylation of PLC-gamma 1. Treatment of Jurkat cells with PMA or forskolin stimulated the phosphorylation of PLC-gamma 1 at serine 1248. PMA treatment also elicited the phosphorylation of PLC-gamma 1 at an unidentified serine site. Phosphopeptide map analysis indicated that the sites of PLC-gamma 1 phosphorylated in Jurkat cells treated with PMA and forskolin are the same as those phosphorylated in vitro by protein kinase C (PKC) and cAMP-dependent protein kinase (PKA), respectively. Stimulation of Jurkat cells with antibodies to CD3 also elicited phosphorylation of PLC-gamma 1 at serine 1248 and at the unidentified serine site phosphorylated in PLC-gamma 1 from PMA-treated cells. Thus, phosphorylation of PLC-gamma 1 by PKC or PKA at serine 1248 may modulate the interaction of PLC-gamma 1 with the protein tyrosine kinase or the protein tyrosine phosphatase; this altered interaction may, at least in part, be responsible for the decreased tyrosine phosphorylation of PLC-gamma 1 seen in PMA- and forskolin-treated Jurkat cells. Furthermore, in the absence of PMA, activation of PKC by diacylglycerol provides a negative feedback signal responsible for reducing the phosphotyrosine contents of PLC-gamma 1.     

Protein kinase C regulates MARCKS cycling between the plasma membrane and lysosomes in fibroblasts.

MARCKS is a protein kinase C (PKC) substrate that is phosphorylated during neurosecretion, phagocyte activation and growth factor-dependent mitogenesis. MARCKS binds calcium/calmodulin and crosslinks F-actin, and both these activities are regulated by PKC-dependent phosphorylation. We present evidence here that PKC-dependent phosphorylation also regulates the cycling of MARCKS between the plasma membrane and Lamp-1-positive lysosomes. Immuno-fluorescence and immunoelectron microscopy, and subcellular fractionation, demonstrated that MARCKS was predominantly associated with the plasma membrane of resting fibroblasts. Activation of PKC resulted in MARCKS phosphorylation and its displacement from the plasma membrane to Lamp-1-positive lysosomes. MARCKS phosphorylation is required for its translocation to lysosomes since mutating either the serine residues phosphorylated by PKC (phos-) or the PKC inhibitor staurosporine, prevented MARCKS phosphorylation, its release from the plasma membrane, and its subsequent association with lysosomes. In the presence of lysosomotropic agents or nocodazole, MARCKS accumulated on lysosomes and returned to the plasma membrane upon drug removal, further suggesting that the protein cycles between the plasma membrane and lysosomes. In contrast to wild-type MARCKS, the phos- mutant did not accumulate on lysosomes in cells treated with NH4Cl, suggesting that basal phosphorylation of MARCKS promotes its constitutive cycling between these two compartments.     

Phosphorylation-dependent binding of a synthetic MARCKS peptide to calmodulin

A 25-amino acid peptide, containing the four protein kinase C (PKC) phosphorylation sites and the calmodulin (CaM) binding domain of the myristoylated alanine-rich C kinase substrate (MARCKS) protein, has been synthesized and used to determine the effects of phosphorylation on its binding and regulation of CaM. PKC phosphorylation of this peptide (3.0 mol of Pi/mol of peptide) produced a 200-fold decrease in its affinity for CaM. PKC phosphorylation of the peptide resulted in its dissociation from CaM over a time course that paralleled the phosphorylation of 1 mol of serine/mol of peptide. The peptide inhibited CaM's binding to myosin light chain kinase and CaM's stimulation of phosphodiesterase and calcineurin. PKC phosphorylation of the peptide resulted in a rapid release of bound CaM, allowing its subsequent binding to myosin light chain kinase (t1/2 = 1.6 min), stimulation of phosphodiesterase (t1/2 = 1.2 min) and calcineurin (t1/2 = 1.7 min). Partially purified MARCKS protein produced a similar inhibition of CaM-phosphodiesterase which was reversed by PKC phosphorylation. PKC phosphorylation of the peptide occurred primarily at serine 8 and serine 12, and phosphorylation of serine 12 regulated peptide affinity for CaM. Thus, PKC phosphorylation of the peptide and the MARCKS protein results in the rapid release of CaM and the subsequent activation of CaM-dependent enzymes. This process might allow for interplay between PKC and CaM-dependent signal transduction pathways.     

Inhibitory phosphorylation of PP1alpha catalytic subunit during the G(1)/S transition.

We have shown earlier that, in cells expressing the retinoblastoma protein (pRB), a protein phosphatase (PP) 1alpha mutant (T320A) resistant to inhibitory phosphorylation by cyclin-dependent kinases (Cdks) causes G(1) arrest. In this study, we examined the cell cycle-dependent phosphorylation of PP1alpha in vivo using three different antibodies. PP1alpha was phosphorylated at Thr-320 during M-phase and again in late G(1)- through early S-phase. Inhibition of Cdk2 led to a small increase in PP1 activity and also prevented PP1alpha phosphorylation. In vitro, PP1alpha was a substrate for Cdk2 but not Cdk4. In pRB-deficient cells, phosphorylation of PP1alpha occurred in M-phase but not at G(1)/S. G(1)/S phosphorylation was at least partially restored after reintroduction of pRB into these cells. Consistent with this result, PP1alpha phosphorylated at Thr-320 co-precipitated with pRB during G(1)/S but was found in extracts immunodepleted of pRB in M-phase. In conjunction with earlier studies, these results indicate that PP1alpha may control pRB function throughout the cell cycle. In addition, our new results suggest that different subpopulations of PP1alpha regulate the G(1)/S and G(2)/M transitions and that PP1alpha complexed to pRB requires inhibitory phosphorylation by G(1)-specific Cdks in order to prevent untimely reactivation of pRB and permit transition from G(1)- to S-phase and/or complete S-phase.     

Regulation of phosphorylation at Ser of GluN2B receptor in the postsynaptic density

Neuronal N-methyl-D-aspartate subtype of ionotropic glutamate receptor (NMDAR) that plays essential roles in excitatory synaptic transmission is regulated by phosphorylation. However, the kinases and phosphatases involved in this regulation are not completely known. We show that the GluN2B subunit of NMDAR is phosphorylated at Ser¹³⁰³ by protein kinase C (PKC) and is dephosphorylated by protein phosphatase 1 (PP1), but not protein phosphatase 2A (PP2A) in isolated postsynaptic density (PSD). Although PSD is known to harbor PKC, PP1 and PP2A, their ability to regulate phosphorylation of GluN2B-Ser¹³⁰³ would depend on the accessibility of GluN2B-Ser¹³⁰³ to these proteins. Since PSD preparation is likely to maintain the organization of its component proteins as inside neurons, accessibility of kinases and phosphatases to GluN2B-Ser¹³⁰³in vivo would be addressed by experiments using this system. Using an antibody specific for the phosphorylated state of GluN2B-Ser¹³⁰³ we demonstrate that PP1 is the major phosphatase in rat brain PSD that can dephosphorylate the GluN2B-Ser¹³⁰³ endogenous to PSD. We also show that PKC present in PSD can phosphorylate GluN2B-Ser¹³⁰³. The events reported here might be important in regulating GluN2B-Ser¹³⁰³ phosphorylation in vivo.     

Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5

The serine/threonine protein phosphatase protein phosphatase 1 (PP1) is known to play an important role in learning and memory by mediating local and downstream aspects of synaptic signaling, but how PP1 activity is controlled in different forms of synaptic plasticity remains unknown. We find that synaptic N-methyl-d-aspartate (NMDA) receptor stimulation in neurons leads to activation of PP1 through a mechanism involving inhibitory phosphorylation at Thr320 by Cdk5. Synaptic stimulation led to proteasome-dependent degradation of the Cdk5 regulator p35, inactivation of Cdk5, and increased auto-dephosphorylation of Thr320 of PP1. We also found that neither inhibitor-1 nor calcineurin were involved in the control of PP1 activity in response to synaptic NMDA receptor stimulation. Rather, the PP1 regulatory protein, inhibitor-2, formed a complex with PP1 that was controlled by synaptic stimulation. Finally, we found that inhibitor-2 was critical for the induction of long-term depression in primary neurons. Our work fills a major gap regarding the regulation of PP1 in synaptic plasticity.     

Thrombin-induced phosphorylation of MARCKS does not alter its interactions with calmodulin or actin

Myristoylated alanine-rich C kinase substrate (MARCKS) is a calmodulin (CaM)- and actin-binding protein and prominent protein kinase C (PKC) substrate. In vitro phosphorylation of MARCKS by PKC has been shown to induce the release of both CaM and actin, leading to the suggestion that MARCKS may regulate CaM availability during agonist-induced signalling. In support of this hypothesis we previously demonstrated that thrombin-induced MARCKS phosphorylation in endothelial cells (EC) parallels activation of myosin light chain kinase, a CaM-dependent enzyme. To test this theory further, we transfected CHO cells, which normally do not express significant levels of MARCKS, with a MARCKS cDNA. The thrombin-stimulated phosphorylation of myosin light chains and the sensitivity to CaM antagonists in the MARCKS overexpressing cells was the same as that in control CHO cells. MARCKS associated with the actin cytoskeleton in EC was markedly increased upon treatment with the PKC activator, PMA, but only modestly enhanced by thrombin treatment. Similarly, colocalisation of MARCKS with actin was enhanced when the EC were challenged with PMA but not thrombin. These data may be partially explained by PKC-independent phosphorylation of MARCKS in response to thrombin stimulation.     

Cdk5 phosphorylation of EFhd2 at S74 affects its calcium binding activity

EFhd2 is a calcium binding protein, which is highly expressed in the central nervous system and associated with pathological forms of tau proteins in tauopathies. Previous phosphoproteomics studies and bioinformatics analysis suggest that EFhd2 may be phosphorylated. Here, we determine whether Cdk5, a hyperactivated kinase in tauopathies, phosphorylates EFhd2 and influence its known molecular activities. The results indicated that EFhd2 is phosphorylated by brain extract of the transgenic mouse CK-p25, which overexpresses the Cdk5 constitutive activator p25. Consistently, in vitro kinase assays demonstrated that Cdk5, but not GSK3β, directly phosphorylates EFhd2. Biomass, tandem mass spectrometry, and mutagenesis analyses indicated that Cdk5 monophosphorylates EFhd2 at S74, but not the adjacent S76. Furthermore, Cdk5-mediated phosphorylation of EFhd2 affected its calcium binding activity. Finally, a phospho-specific antibody was generated against EFhd2 phosphorylated at S74 and was used to detect this phosphorylation event in postmortem brain tissue from Alzheimer''s disease and normal-aging control cases. Results demonstrated that EFhd2 is phosphorylated in vivo at S74. These results imply that EFhd2''s physiological and/or pathological function could be regulated by its phosphorylation state.     

Downregulation of PP2A(Cdc55) phosphatase by separase initiates mitotic exit in budding yeast

After anaphase, the high mitotic cyclin-dependent kinase (Cdk) activity is downregulated to promote exit from mitosis. To this end, in the budding yeast S. cerevisiae, the Cdk counteracting phosphatase Cdc14 is activated. In metaphase, Cdc14 is kept inactive in the nucleolus by its inhibitor Net1. During anaphase, Cdk- and Polo-dependent phosphorylation of Net1 is thought to release active Cdc14. How Net1 is phosphorylated specifically in anaphase, when mitotic kinase activity starts to decline, has remained unexplained. Here, we show that PP2A(Cdc55) phosphatase keeps Net1 underphosphorylated in metaphase. The sister chromatid-separating protease separase, activated at anaphase onset, interacts with and downregulates PP2A(Cdc55), thereby facilitating Cdk-dependent Net1 phosphorylation. PP2A(Cdc55) downregulation also promotes phosphorylation of Bfa1, contributing to activation of the "mitotic exit network" that sustains Cdc14 as Cdk activity declines. These findings allow us to present a new quantitative model for mitotic exit in budding yeast.     

Phosphorylation at serine 75 is required for UV-mediated degradation of human Cdc25A phosphatase at the S-phase checkpoint

The human Cdc25A phosphatase plays a pivotal role at the G1/S transition by activating cyclin E and A/Cdk2 complexes through dephosphorylation. In response to ionizing radiation, Cdc25A is phosphorylated by both Chk1 and Chk2 on Ser-123. This in turn leads to ubiquitylation and rapid degradation of Cdc25A by the proteasome resulting in cell cycle arrest. We found that in response to UV irradiation, Cdc25A is phosphorylated at a different serine residue, Ser-75. Significantly, Cdc25A mutants carrying alanine instead of either Ser-75 or Ser-123 demonstrate that only Ser-75 mediates protein stabilization in response to UV-induced DNA damage. As a consequence, cyclin E/Cdk2 kinase activity was high. Furthermore, we find that Cdc25A was phosphorylated by Chk1 on Ser-75 in vitro and that the same site was also phosphorylated in vivo. Taken together, these data strongly suggest that phosphorylation of Cdc25A on Ser-75 by Chk1 and its subsequent degradation is required to delay cell cycle progression in response to UV-induced DNA lesions.     

Cdk1 phosphorylation of fission yeast paxillin inhibits its cytokinetic ring localization

Divisions of the genetic material and cytoplasm are coordinated spatially and temporally to ensure genome integrity. This coordination is mediated in part by the major cell cycle regulator cyclin-dependent kinase (Cdk1). Cdk1 activity peaks during mitosis, but during mitotic exit/cytokinesis Cdk1 activity is reduced, and phosphorylation of its substrates is reversed by various phosphatases including Cdc14, PP1, PP2A, and PP2B. Cdk1 is known to phosphorylate several components of the actin- and myosin-based cytokinetic ring (CR) that mediates division of yeast and animal cells. Here we show that Cdk1 also phosphorylates the Schizosaccharomyces pombe CR component paxillin Pxl1. We determined that both the Cdc14 phosphatase Clp1 and the PP1 phosphatase Dis2 contribute to Pxl1 dephosphorylation at mitotic exit, but PP2B/calcineurin does not. Preventing Pxl1 phosphorylation by Cdk1 results in increased Pxl1 levels, precocious Pxl1 recruitment to the division site, and increased duration of CR constriction. In vitro Cdk1-mediated phosphorylation of Pxl1 inhibits its interaction with the F-BAR domain of the cytokinetic scaffold Cdc15, thereby disrupting a major mechanism of Pxl1 recruitment. Thus, Pxl1 is a novel substrate through which S. pombe Cdk1 and opposing phosphatases coordinate mitosis and cytokinesis.     

Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes.

Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) was compared with calcium/phosphatidylserine (Ca/PS). The substrate specificity of PKC was more limited with PS/PMA. Substrates could be divided into three overlapping groups according to their relative level of phosphorylation: C1, relatively preferred substrates with Ca/PS, included dephosphin, histone, and peptide GS1-10. C2, relatively preferred with PS/PMA, included myelin basic protein and MARCKS. C3, substrates independent of activators. PS/PMA altered the Vmax of PKC for substrate, and decreased the Km for Mg2+. Differential substrate phosphorylation by PS/PMA also occurred for PKC isozymes resolved by hydroxylapatite chromatography and was most dramatic for PKC-alpha, which could no longer phosphorylate histone or GS1-12. Differential activities of PKC were also observed in synaptosol and in intact synaptosomes where PMA stimulated phosphorylation of MARCKS, but not dephosphin. It was further shown that dephosphin was indeed a substrate of PKC in the intact synaptosomes by use of a repolarization-dependent dephosphin phosphorylation assay. The differential PKC activities could also be distinguished by inhibitors. H-7 was equipotent, palmitoylcarnitine did not inhibit in vitro C2 phosphorylation, but inhibited dephosphin in intact synaptosomes, and sphingosine did not inhibit C1 substrates and was without effect on dephosphin in intact synaptosomes. Therefore PS/PMA alters or limits the substrate specificity of PKC, leading to a differential substrate phosphorylation in vitro and in intact synaptosomes and differential inhibitor sensitivity. The pattern of protein phosphorylation observed after PKC activation in intact cells will therefore be dependent upon the activator.     

Characteristics of the F52 protein, a MARCKS homologue.

A recently cloned mouse cDNA designated F52 encodes a putative protein with striking sequence similarity to the MARCKS protein, a major cellular substrate for protein kinase C (PKC). Major regions of sequence similarity include the amino-terminal myristoylation consensus sequence and the central calmodulin-binding/PKC phosphorylation site domain. The F52 protein was expressed in Escherichia coli with apparent M(r) 50,000; it was a substrate for PKC and comigrated on two-dimensional electrophoresis with a myristoylated protein whose phosphorylation was stimulated by phorbol 12-myristate 13-acetate in mouse neuroblastoma cells. The F52 protein also was myristoylated in E. coli by co-expression with N-myristoyltransferase. A 24-amino acid peptide derived from the protein's phosphorylation site domain was a good substrate for PKC; like the cognate MARCKS peptide, it was phosphorylated with high affinity (S0.5 = 173 nM) and positive cooperativity (KH = 5.4). The F52 peptide also bound calmodulin with high affinity (Kd = less than 3 nM); this binding could be disrupted by phosphorylation of the peptide with PKC, with a half-time of 8 min. The F52 protein is clearly a member of the MARCKS family as defined by primary sequence; in addition, the two proteins share several key attributes that may be functionally important.     

Protein kinase C inhibits the transplasma membrane influx of Ca2+ triggered by 4-aminopyridine in Jurkat T lymphocytes

4-aminopyridine (4AP) is a general blocker of voltage-dependent K+ channels. This pyridine derivative has also been shown to inhibit T cell proliferation, to modulate immune responses and to alleviate some of the symptoms associated with neurological disorders such as multiple sclerosis, myasthenia gravis and Alzheimer's disease. 4AP triggers a Ca2+ response in lymphocytes, astrocytes, neurons and muscle cells but little is known about the regulation of the 4AP response in these cells. We report that 4AP induced a non-capacitative transplasma membrane influx of Ca2+ in Jurkat T lymphocytes. The influx of Ca2+ was not affected by activation or inhibition of protein kinase A (PKA). In contrast, activation of protein kinase C (PKC) by phorbol myristyl acetate (PMA), mezerein or 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited the influx of Ca2+ triggered by 4AP. The inhibitory effect of PKC could be prevented by prior exposure of the cells to the PKC inhibitor GF 109203X. Under these conditions, mezerein and OAG no longer inhibited the 4AP-dependent Ca2+ response. Inhibition of serine and threonine protein phosphatases PP1 and PP2A by treating the cells with calyculin A (CalA) reduced the Ca2+ response to 4AP. Okadaic acid (OA) had no effect, suggesting an involvement of PP1. A combination of CalA and OAG (or PMA) abolished the influx of Ca2+ induced by 4AP, adding further evidence to the importance of protein phosphorylation in the modulation of the 4AP response. Our data suggest that the transplasma membrane influx of Ca2+ triggered by 4AP in Jurkat T cells can be modulated by the opposite actions of PKC and protein serine and threonine phosphatase(s).