Myristoylated, alanine-rich C-kinase substrate phosphorylation regulates growth cone adhesion and pathfinding

Repellents evoke growth cone turning by eliciting asymmetric, localized loss of actin cytoskeleton together with changes in substratum attachment. We have demonstrated that semaphorin-3A (Sema3A)-induced growth cone detachment and collapse require eicosanoid-mediated activation of protein kinase C epsilon (PKC epsilon) and that the major PKC epsilon target is the myristoylated, alanine-rich C-kinase substrate (MARCKS). Here, we show that PKC activation is necessary for growth cone turning and that MARCKS, while at the membrane, colocalizes with alpha3-integrin in a peripheral adhesive zone of the growth cone. Phosphorylation of MARCKS causes its translocation from the membrane to the cytosol. Silencing MARCKS expression dramatically reduces growth cone spread, whereas overexpression of wild-type MARCKS inhibits growth cone collapse triggered by PKC activation. Expression of phosphorylation-deficient, mutant MARCKS greatly expands growth cone adhesion, and this is characterized by extensive colocalization of MARCKS and alpha3-integrin, resistance to eicosanoid-triggered detachment and collapse, and reversal of Sema3A-induced repulsion into attraction. We conclude that MARCKS is involved in regulating growth cone adhesion as follows: its nonphosphorylated form stabilizes integrin-mediated adhesions, and its phosphorylation-triggered release from adhesions causes localized growth cone detachment critical for turning and collapse.     

Differential Changes in the Phosphorylation of the Protein Kinase C Substrates Myristoylated Alanine-Rich C Kinase Substrate and Growth-Associated Protein-43/B-50 Following Schaffer Collateral Long-Term Potentiation and Long-Term Depression

Activation of protein kinase C (PKC) is one of the biochemical pathways thought to be activated during activity-dependent synaptic plasticity in the brain, and long-term potentiation (LTP) and long-term depression (LTD) are two of the most extensively studied models of synaptic plasticity. Here we have examined changes in the in situ phosphorylation level of two major PKC substrates, myristoylated alanine-rich C kinase substrate (MARCKS) and growth-associated protein (GAP)-43/B-50, after pharmacological stimulation or induction of LTP or LTD in the CA1 field of the hippocampus. We find that direct PKC activation with phorbol esters, K+-induced depolarization, and activation of metabotropic glutamate receptors increase the in situ phosphorylation of both MARCKS and GAP-43/B-50. The induction of LTP increased the in situ phosphorylation of both MARCKS and GAP-43/B-50 at 10 min following high-frequency stimulation, but only GAP-43/B-50 phosphorylation remained elevated 60 min after LTP induction. Furthermore, blockade of LTP induction with the NMDA receptor antagonist D-2-amino-5-phosphonopentanoic acid prevented elevations in GAP-43/B-50 phosphorylation but did not prevent the elevation in MARCKS phosphorylation 10 min following LTP induction. The induction of LTD resulted in a reduction in GAP-43/B-50 phosphorylation but did not affect MARCKS phosphorylation. Together these findings show that activity-dependent synaptic plasticity elicits PKC-mediated phosphorylation of substrate proteins in a highly selective and coordinated manner and demonstrate the compartmentalization of PKC-substrate interactions. Key Words: Protein kinase C-Myristoylated alanine-rich C kinase substrate-Growth-associated protein-43-Long-term potentiation-Long-term depression-(RS)-alpha-Methyl-4-carboxyphenylglycine-D-2-Amino-5-ph osphonopentanoic acid-Glutamate.     

Genetic depletion of brain 5HT reveals a common molecular pathway mediating compulsivity and impulsivity

The repellent semaphorin 3A (Sema3A) causes growth cone turning or collapse by triggering cytoskeletal rearrangements and detachment of adhesion sites. Growth cone detachment is dependent on eicosanoid activation of protein kinase C epsilon (PKCε), but the characterization of the phospholipase A(2) (PLA(2) ) that releases arachidonic acid (AA) for eicosanoid synthesis has remained elusive. Here, we show, in rat dorsal root ganglion (DRG) neurons, that Sema3A stimulates PLA(2) activity, that Sema3A-induced growth cone turning and collapse are dependent on the release of AA, and that the primary PLA(2) involved is the group IV α isoform (GIVA). Silencing GIVA expression renders growth cones resistant to Sema3A-induced collapse, and GIVA inhibition reverses Sema3A-induced repulsion into attraction. These studies identify a novel, early step in Sema3A-signaling and a PLA(2) necessary for growth cone repulsion and collapse.     

Protein kinase C alpha phosphorylates and negatively regulates diacylglycerol kinase zeta

Diacylglycerol kinase (DGK) terminates diacylglycerol (DAG) signaling by phosphorylating DAG to produce phosphatidic acid, which also has signaling properties. Thus, precise control of DGK activity is essential for proper signal transduction. We demonstrated previously that a peptide corresponding to the myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation site domain (PSD) in DGK zeta was phosphorylated in vitro by an active fragment of protein kinase C (PKC). In the present study, we tested full-length DGK zeta and found that PKC alpha phosphorylated DGK zeta on serines within the MARCKS PSD in vitro and in vivo. DGK zeta also coimmunoprecipitated with PKC alpha, suggesting that they reside in a regulated signaling complex. We then tested whether phosphorylation affected DAG kinase activity. We found that a mutant (DGK zeta S/D) in which serines within the MARCKS PSD were altered to aspartates (to mimic phosphorylation) had lower activity compared with wild-type DGK zeta or a control mutant (DGK zeta S/N) in which the same serines were changed to asparagines. Furthermore, activation of PKC alpha by phorbol 12-myristate 13-acetate inhibited the activity of wild-type DGK zeta, but not DGK zeta S/D, in human embryonic kidney 293 cells. These results suggest that by phosphorylating the MARCKS PSD, PKC alpha attenuates DGK zeta activity. Supporting this, we found that cells expressing DGK zeta S/D had higher DAG levels and grew more rapidly compared with cells expressing DGK zeta S/N that could not be phosphorylated. Taken together, these results indicate that PKC alpha phosphorylates DGK zeta in cells, and this phosphorylation inhibits its kinase activity to remove cellular DAG, thereby affecting cell growth.     

Thrombin and histamine rapidly stimulate the phosphorylation of the myristoylated alanine-rich C-kinase substrate in human umbilical vein endothelial cells: evidence for distinct patterns of protein kinase activation.

Human alpha-thrombin and histamine each stimulates protein phosphorylation in human umbilical vein endothelial cells (HUVEC). We have identified the most prominent of these phosphoproteins by immunoprecipitation as the human homolog of the widely distributed myristoylated alanine-rich C-kinase substrate (MARCKS). Stimulation by 0.1-10 U/ml of alpha-thrombin produces a time-dependent, sustained (plateau 3-5 min) level of MARCKS phosphorylation. MARCKS phosphorylation requires thrombin catalytic activity but not receptor binding and is also seen in response to stimulation by a peptide, TR (42-55), that duplicates a portion of the thrombin receptor tethered ligand created by thrombin proteolytic activity. One micromolar histamine, like alpha-thrombin, produces sustained phosphorylation of MARCKS (plateau 3-5 min). In contrast, 100 microM histamine results in rapid but transient MARCKS phosphorylation (peak 1-3 min). HUVEC treated with 100 microM histamine for 5 min can be restimulated by alpha-thrombin but not fresh histamine, suggesting that the histamine receptor was desensitized. MARCKS phosphorylation can also be induced by several exogenous protein kinase C (PKC) activators and both alpha-thrombin- and histamine-induced MARCKS phosphorylation are inhibited by the PKC antagonist staurosporine. However, while prolonged PMA pretreatment ablates histamine-induced MARCKS phosphorylation, the ability of thrombin to induce MARCKS phosphorylation is retained. These findings provide evidence for agonist-specific pathways of protein kinase activation in response to thrombin and histamine in HUVEC.     

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.     

PKC phosphorylates MARCKS Ser159 not only directly but also through RhoA/ROCK

It is well recognized that phorbol 12,13-dibutyrate (PDBu)-activated PKC directly phosphorylates myristoylated alanine-rich C kinase substrate (MARCKS), whose phosphorylation is used as a marker of PKC activation. However, in SH-SY5Y neuroblastoma cells, Western blotting analyses revealed that Rho-associated coiled-coil kinase (ROCK)-specific inhibitor H-1152 inhibited PDBu-induced phosphorylation, and that a small G-protein inhibitor, toxin B, also inhibited MARCKS phosphorylation. Furthermore, in GST pull-down assays, PDBu induced RhoA activation in SH-SY5Y cells, and this activation was inhibited by PKC inhibitor Ro-31-8220. Finally, we showed that the transfection of a dominant negative form of RhoA inhibited PDBu-induced MARCKS phosphorylation in immunocytochemistries. These findings suggest that some PDBu-induced MARCKS phosphorylation includes the RhoA/ROCK pathway in SH-SY5Y cells.     

Activation of Ras, Raf-1 and protein kinase C in differentiating human neuroblastoma cells after treatment with phorbolester and NGF

The human neuroblastoma cell line SH-SY5Y/TrkA differentiates in vitro and acquires a sympathetic phenotype in response to phorbolester (activator of protein kinase C, PKC) in the presence of serum or growth factors, or nerve growth factor (NGF). We have now investigated to what extent phorbolester and NGF cause activation of Ras and Raf-1 and the involvement of PKC in this response in differentiating SH-SY5Y/TrkA cells. NGF stimulated increased accumulation of Ras-GTP and a threefold activation of Raf-1. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect on the amount of Ras-GTP but led to a smaller activation of Raf-1. NGF caused a limited increase in phosphorylation of Raf-1 compared with TPA, and NGF-induced Raf activity was independent of PKC. Analysis of phosphorylation of the endogenous PKC substrate myristoylated alanine-rich C-kinase substrate (MARCKS), and of subcellular distribution of PKC-alpha, -delta, and -epsilon revealed that NGF only caused a very small activation of PKC in SH-SY5Y/TrkA cells. The results identify Raf-1 as a target for both TPA- and NGF-induced signals in differentiating SH-SY5Y/TrkA cells and demonstrate that signalling to Raf-1 was mediated via distinct mechanisms.     

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.     

Glutamate-induced protein phosphorylation in cerebellar granule cells: Role of protein kinase C

Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells.³²P-labelled cells have been stimulated with 100 M glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells.Abbreviations (NMDA) N-methyl-D-aspartate - (PKC) protein kinase C - (EAA) excitatory aminoacids - (GAMSA) -D-glutamylaminomethylsulfonate - (MK801) (+)-10,11-dihydro-5-methyl-5-H-dibenzo-(a,d)-cyclohepten-5,10imine - (TPA) phorbol 12-myristate 13-acetate - (MARCKS) myristoylated alanine-rich C kinase substrate - (GAP-43) growth-associated protein-43 - (SDS) sodium dodecyl sulfate - (PAGE) polyacrylamide gel electrophoresis - (H7) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - (DIV) days in vitro     

Characterization of a selective protein kinase C substrate derived from the MARCKS phosphorylation site domain for use in brain tissue homogenates

Protein kinase C (PKC) isozymes play crucial roles in neuronal signal transduction and can regulate transmitter release, ion channels, neural development, and plasticity. In vitro assays of PKC are frequently used to associate PKC activity with cellular function, and the availability of selective PKC substrates can facilitate such studies. We have characterized a commercially available 12 amino acid peptide derived from the myristoylated alanine-rich C kinase substrate (MARCKS-PSD, Calbiochem) for use in crude rat brain homogenates. Assays were performed at 25 degrees C for 10 min (linear up to 12 min) using optimal concentrations of calcium and lipid cofactors. Kinetic analysis of MARCKS-PSD phosphorylation by PKC purified from rat brain gave a K(m) of 2.3 microM, which was similar to the K(m) of 2.8 microM obtained using rat brain cortical homogenates. The selective PKC inhibitor bisindolylmaleimide reduced phosphorylation of MARCKS-PSD in a concentration-dependent manner, with greater than 95% inhibition at 1.0 microM. MARCKS-PSD was more potent than another widely used selective PKC substrate (neurogranin((28-43)) and was a good substrate for human recombinant PKC alpha, delta, and epsilon but not zeta. The ontogeny of PKC activity was examined in the cortex and cerebellum. PKC activity was low at birth and reached adult levels by 21 days of age in both regions. Calcium-independent PKC activity in brain homogenates could be measured with MARCKS-PSD and accounted for approximately 25 and 10% of total activity in 1-day-old and adult rat cortex, respectively. These results suggest that the MARCKS-PSD peptide can be used as a selective PKC substrate in rat brain homogenates.     

Isoform-specific phosphorylation of metabotropic glutamate receptor 5 by protein kinase C (PKC) blocks Ca2+ oscillation and oscillatory translocation of Ca2+-dependent PKC

Prolonged activation of metabotropic glutamate receptor 5a (mGluR5a) causes synchronized oscillations in intracellular calcium, inositol 1,4,5-trisphosphate production, and protein kinase C (PKC) activation. Additionally, mGluR5 stimulation elicited cyclical translocations of myristoylated alanine-rich protein kinase C substrate, which were opposite to that of gammaPKC (i.e. from plasma membrane to cytosol) and dependent on PKC activity, indicating that myristoylated alanine-rich protein kinase C substrate is repetitively phosphorylated by oscillating gammaPKC on the plasma membrane. Mutation of mGluR5 Thr(840) to aspartate abolished the oscillation of gammaPKC, but the mutation to alanine (T840A) did not. Cotransfection of gammaPKC with betaIIPKC, another Ca2+-dependent PKC, resulted in synchronous oscillatory translocation of both classical PKCs. In contrast, cotransfection of deltaPKC, a Ca2+-independent PKC, abolished the oscillations of both gammaPKC and inositol 1,4,5-trisphosphate. Regulation of the oscillations was dependent on deltaPKC kinase activity but not on gammaPKC. Furthermore, the T840A-mGluR5-mediated oscillations were not blocked by the deltaPKC overexpression. These results revealed that activation of mGluR5 causes translocation of both gammaPKC and deltaPKC to the plasma membrane. deltaPKC, but not gammaPKC, phosphorylates mGluR5 Thr(840), leading to the blockade of both Ca2+ oscillations and gammaPKC cycling. This subtype-specific targeting proposes the molecular basis of the multiple functions of PKC.     

Decoding of short-lived Ca2+ influx signals into long term substrate phosphorylation through activation of two distinct classes of protein kinase C

In electrically excitable cells, membrane depolarization opens voltage-dependent Ca(2+) channels eliciting Ca(2+) influx, which plays an important role for the activation of protein kinase C (PKC). However, we do not know whether Ca(2+) influx alone can activate PKC. The present study was conducted to investigate the Ca(2+) influx-induced activation mechanisms for two classes of PKC, conventional PKC (cPKC; PKCalpha) and novel PKC (nPKC; PKCtheta), in insulin-secreting cells. We have demonstrated simultaneous translocation of both DsRed-tagged PKCalpha to the plasma membrane and green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate to the cytosol as a dual marker of PKC activity in response to depolarization-evoked Ca(2+) influx in the DsRed-tagged PKCalpha and GFP-tagged myristoylated alanine-rich C kinase substrate co-expressing cells. The result indicates that Ca(2+) influx can generate diacylglycerol (DAG), because cPKC is activated by Ca(2+) and DAG. We showed this in three different ways by demonstrating: 1) Ca(2+) influx-induced translocation of GFP-tagged C1 domain of PKCgamma, 2) Ca(2+) influx-induced translocation of GFP-tagged pleckstrin homology domain, and 3) Ca(2+) influx-induced translocation of GFP-tagged PKCtheta, as a marker of DAG production and/or nPKC activity. Thus, Ca(2+) influx alone via voltage-dependent Ca(2+) channels can generate DAG, thereby activating cPKC and nPKC, whose activation is structurally independent of Ca(2+).     

A continuous fluorescence assay for protein kinase C

A 6-acryloyl-2-dimethylaminonapthalene (acrylodan)-labeled 25-amino acid peptide (acrylodan-CKK-KKRFSFKKSFKLSGFSFKKNKK-COO-), containing the protein kinase C (PKC) phosphorylation sites of brain myristoylated alanine-rich kinase C substrate protein, undergoes a 20% fluorescence decrease when it is phosphorylated by phospholipid/calcium-dependent protein kinase (PKC). This fluorescence decrease is dependent on the presence of PKC, calcium (half-maximal stimulation at pCa = 6.2), phosphatidylserine, diacylglycerol, or phorbol-12-myristate-13-acetate (half-maximal stimulation at 2 nM) and ATP, and correlates well (r = 0.997) with [32P]phosphate incorporation into the peptide. This fluorescence assay allows detection of 0.02 nM PKC, while similar concentrations of cyclic AMP-dependent or type II calmodulin-dependent protein kinases produced no change in peptide fluorescence. The method can be used to assay purified PKC as well as activity in crude brain homogenates. Incubation of PKC with staurosporine inhibits the fluorescence decrease with an IC50 of 2 nM. Thus the fluorescence decrease that occurs in the acrylodan-peptide provides a continuous fluorescence assay for PKC activity.     

Interaction of the C-terminal region of the rat serotonin transporter with MacMARCKS modulates 5-HT uptake regulation by protein kinase C

The serotonin transporter (SERT) mediates the re-uptake of released serotonin into presynaptic nerve terminals. Its activity is regulated by different mechanisms including protein kinase C (PKC) triggered internalization. Here, we used yeast 2-hybrid screening and cotransfection into 293 cells to identify a homologue of the myristoylated alanine-rich C kinase substrate (MARCKS), MacMARCKS, as a C-terminally interacting protein of SERT. Upon cotransfection with SERT, MacMARCKS caused a reduction in the maximal rate of [(3)H]serotonin uptake and reduced its down-regulation elicited by activation of PKC. Our data are consistent with MARCKS proteins regulating the plasma membrane dynamics of neurotransmitter transporters.     

Phosphorylation of the Casein Kinase II Domain of B-50 (GAP-43) in Rat Cortical Growth Cones

Abstract: Growth-associated phosphoprotein B-50 is a neural protein kinase C (PKC) substrate enriched in nerve growth cones that has been implicated in growth cone plasticity. Here we investigated whether B-50 is a physiological substrate for casein kinase II (CKII) in purified rat cortical growth cone preparations. Using site-specific proteolysis and known modulators of PKC, in combination with immunoprecipitation, mass spectrometry, and phosphoamino acid analysis, we demonstrate that endogenous growth cone B-50 is phosphorylated at multiple sites, on both serine and threonine residues. Consistent with previous reports, stimulation of PKC activity increased the phosphorylation of only those proteolytic fragments containing Ser⁴¹. Under basal conditions, however, phosphorylation was predominantly associated with fragments not containing Ser⁴¹. Mass spectrometry of tryptic digests of B-50, which had been immunoprecipitated from untreated growth cones, revealed that in situ phosphorylation occurs within peptides B-50_181–198 and B-50_82–98. These peptides contain the major and minor in vitro CKII phosphosites, respectively. In addition, cyanogen bromide digestion of immunoprecipitated chick B-50 generated a 4-kDa C-terminal B-50 phosphopeptide, confirming that phosphorylation of the CKII domain occurs across evolutionary diverse species. We conclude that B-50 in growth cones is not only a substrate for PKC, but also for CKII.     

Tob-mediated cross-talk between MARCKS phosphorylation and ErbB-2 activation

The biochemical path for the activation of ErbB-2 by PKC activator was investigated in MDA-MB-231 human breast cancer cells. We found that PMA-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) increased its binding with Tob that exerts an anti-proliferative effect through the binding with ErbB-2. The phosphorylation site domain (PSD) of MARCKS was relevant to its interaction with Tob. Decreased binding of Tob with ErbB-2 and subsequent activation of ErbB-2 were observed in MDA-MB-231 cells in response to PMA treatment. The present study proposes that MARCKS phosphorylation by PKC removes Tob from ErbB-2 by increasing its binding affinity with Tob, and thereby activates the ErbB-2 mediated signal transduction.     

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.     

Macrophage-enriched myristoylated alanine-rich C kinase substrate and its phosphorylation is required for the phorbol ester-stimulated diffusion of beta 2 integrin molecules

An early event of beta(2) integrin activation is the increased diffusion rate of this molecule on the cell surface, thereby providing integrin molecules with a better chance to meet the ligands. The activation of protein kinase C (PKC) stimulates integrin diffusion by releasing the cytoskeletal constraint on integrin molecules. We report here that macrophage-enriched myristoylated alanine-rich C kinase substrate (MacMARCKS), a membrane-associated PKC substrate involved in integrin activation, is required for this PKC-stimulated diffusion of integrin molecules. Using the single-particle tracking technique, we observed that the activation of PKC stimulated an 11-fold increase in the diffusion rate of beta(2) integrins in wild type J774 macrophage cells but not in those expressing mutant MacMARCKS. Further evidence is provided from a MacMARCKS-deficient cell line in which phorbol esters failed to stimulate the diffusion of integrin. Transfection of wild type MacMARCKS into these cells restored the rapid diffusion rate of the beta(2) integrins. The phosphorylation of MacMARCKS is important because transfection of a nonphosphorylatable MacMARCKS mutant or the addition of staurosporine eliminates the rapid diffusion rate of integrin. Furthermore, adding cytochalasin D bypasses the MacMARCKS deficiency and stimulates beta(2) integrin diffusion, suggesting that MacMARCKS's involvement in integrin activation is prior or at the site of cytoskeleton. Therefore, we conclude that MacMARCKS is required for releasing the cytoskeletal constraint on integrin molecules during PKC-mediated integrin activation.     

Importance of protein kinase C targeting for the phosphorylation of its substrate, myristoylated alanine-rich C-kinase substrate

We visualized the translocation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in living Chinese hamster ovary-K1 cells using MARCKS tagged to green fluorescent protein (MARCKS-GFP). MARCKS-GFP was rapidly translocated from the plasma membrane to the cytoplasm after the treatment with phorbol ester, which translocates protein kinase C (PKC) to the plasma membrane. In contrast, PKC activation by hydrogen peroxide, which was not accompanied by PKC translocation, did not alter the intracellular localization of MARCKS-GFP. Non-myristoylated mutant of MARCKS-GFP was distributed throughout the cytoplasm, including the nucleoplasm, and was not translocated by phorbol ester or by hydrogen peroxide. Phosphorylation of wild-type MARCKS-GFP was observed in cells treated with phorbol ester but not with hydrogen peroxide, whereas non-myristoylated mutant of MARCKS-GFP was phosphorylated in cells treated with hydrogen peroxide but not with phorbol ester. Phosphorylation of both MARCKS-GFPs reduced the amount of F-actin. These findings revealed that PKC targeting to the plasma membrane is required for the phosphorylation of membrane-associated MARCKS and that a mutant MARCKS existing in the cytoplasm can be phosphorylated by PKC activated in the cytoplasm without translocation but not by PKC targeted to the membrane.