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.     

Protein kinase C substrate and inhibitor characteristics of peptides derived from the myristoylated alanine-rich C kinase substrate (MARCKS) protein phosphorylation site domain.

The phosphorylation sites in the myristoylated alanine-rich C kinase substrate or MARCKS protein consist of four serines contained within a conserved, basic region of 25 amino acids, termed the phosphorylation site domain. A synthetic peptide comprising this domain was phosphorylated by both protein kinase C and its catalytic fragment with high affinity and apparent positive cooperativity. Tryptic phosphopeptides derived from the peptide appeared similar to phosphopeptides derived from the phosphorylated intact protein. The peptide was phosphorylated by cAMP- and cGMP-dependent protein kinases with markedly lower affinities. In peptides containing only one of the four serines, with the other three serines replaced by alanine, the affinities for protein kinase C ranged from 25 to 60 nM with Hill constants between 1.8 and 3.0. The potential pseudosubstrate peptide, in which all four serines were replaced by alanines, inhibited protein kinase C phosphorylation of histone or a peptide substrate with an IC50 of 100-200 nM with apparently non-competitive kinetics; it also inhibited the catalytic fragment of protein kinase C with a Ki of 20 nM, with kinetics of the mixed type. The peptide did not significantly inhibit the cAMP- and cGMP-dependent protein kinases. It inhibited Ca2+/calmodulin-dependent protein kinases I, II, and III by competing with the kinases for calmodulin. In addition, the peptide inhibited the Ca2+/calmodulin-independent activity of a proteolytic fragment of Ca2+/calmodulin protein kinase II, with an IC50 approximately 5 microM. Thus, the phosphorylation site domain peptide of the MARCKS protein is a high affinity substrate for protein kinase C in vitro; the cognate peptide containing no serines is a potent but not completely specific inhibitor of both protein kinase C and its catalytic fragment.     

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.     

Ser1928 is a common site for Cav1.2 phosphorylation by protein kinase C isoforms

Voltage-dependent Ca(2+) channel (Ca(v)1.2, L-type Ca(2+) channel) function is highly regulated by hormones and neurotransmitters in large part through the activation of kinases and phosphatases. Regulation of Ca(v)1.2 by protein kinase C (PKC) is of significant physiologic importance, mediating, in part, the cardiac response to hormonal regulation. Although PKC has been reported to mediate activation and/or inhibition of Ca(v)1.2 function, the molecular mechanisms mediating the response have not been definitively elucidated. We show that PKC forms a macromolecular complex with the alpha(1c) subunit of Ca(v)1.2 through direct interaction with the C terminus. This interaction leads to phosphorylation of the channel in response to activators of PKC. We identify Ser(1928) as the residue that is phosphorylated by PKC in vitro and in vivo. Ser(1928) has been identified previously as the site mediating, in part, the protein kinase A up-regulation of channel activity. Thus, the protein kinase A and PKC signaling pathways converge on the Ca(v)1.2 complex at Ser(1928) to increase channel activity. Our results identify two mechanisms leading to regulation of Ca(v)1.2 activity by PKC: pre-association of the channel with PKC isoforms and phosphorylation of specific sites within the alpha(1c) subunit.     

Identification of the protein kinase C phosphorylation site in neuromodulin

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin [Alexander, K. A., Cimler, B. M., Meier, K. E., & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113], and we have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar Km values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [gamma-32P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32P-labeled tryptic peptide was generated from phosphorylated neuromodulin. The sequence of this peptide was IQASFR. The serine in this peptide corresponds to position 41 of the entire protein, which is adjacent to or contained within the calmodulin binding domain of neuromodulin. A synthetic peptide, QASFRGHITRKKLKGEK, corresponding to the calmodulin binding domain with a few flanking residues, including serine-41, was also phosphorylated by protein kinase C. We conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmodulin to neuromodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel tyrosine phosphorylation site in protein kinase D contributes to oxidative stress-mediated activation

Protein kinase D1 (PKD1) is a mediator of oxidative stress signaling where it regulates cellular detoxification and survival. Critical for the regulation of PKD1 activity in response to oxidative stress are Src- and Abl-mediated tyrosine phosphorylations that eventually lead to protein kinase Cdelta (PKCdelta)-mediated activation of PKD1. Here we identify Tyr95 in PKD1 as a previously undescribed phosphorylation site that is regulated by oxidative stress. Our data suggest that PKD1 phosphorylation at Tyr95 generates a binding motif for PKCdelta, and that oxidative stress-mediated PKCdelta/PKD interaction results in PKD1 activation loop phosphorylation and activation. We further analyzed all PKD isoforms for this mechanism and show that PKD enzymes PKD1 and PKD2 are targets for PKCdelta in response to oxidative stress, and that PKD3 is not a target because it lacks the relevant tyrosine residue that generates a PKCdelta interaction motif.     

Comparison of an endogenous protein kinase C substrate in rat aorta with rat brain MARCKS

We have compared the properties of a rat aorta-derived protein kinase C substrate (p75) with those of 80 kDa kinase C substrates from rat brain (MARCKS) and rabbit aorta (p80). Rat aortic p75 appeared to be closely related to rat brain MARCKS on the basis of: solubility in perchloric acid and trichloroacetic acid, heat stability, isoelectric point (pI 4.2), overall V8 protease phosphopeptide map, and immunocrossreactivity with an antibody directed against the N-terminal domain of MARCKS. However, p75 could be distinguished from rat brain MARCKS and from the rabbit aorta-derived p80 on the basis of its consistently more rapid electrophoretic mobility in SDS-containing gels, and in terms of a unique proteolytic phosphopeptide found in MARCKS but not in aortic p75. We conclude that p75 probably belongs to the family of protein kinase C substrates represented by MARCKS, and that differences in post-translational processing (glycosylation) or mRNA processing may account for the unique properties of the p75 protein in rat aortic tissue.Abbreviations p75 75,000 Da protein - MARCKS Myristoylated Alanine-Rich C Kinase Substrate     

Membrane translocation of novel protein kinase Cs is regulated by phosphorylation of the C2 domain

Ca(2+)-independent or novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain of unknown function. Removal of the C2 domain of the Aplysia nPKC Apl II allows activation of the enzyme at lower concentrations of phosphatidylserine, suggesting an inhibitory role for the C2 domain in enzyme activation. However, the mechanism for C2 domain-mediated inhibition is not known. Mapping of the autophosphorylation sites for protein kinase C (PKC) Apl II reveals four phosphopeptides in the regulatory domain of PKC Apl II, two of which are in the C2 domain at serine 2 and serine 36. Unlike most PKC autophosphorylation sites, these serines could be phosphorylated in trans. Interestingly, phosphorylation of serine 36 increased binding of the C2 domain to phosphatidylserine membranes in vitro. In cells, PKC Apl II phosphorylation at serine 36 was increased by PKC activators, and PKC phosphorylated at this position translocated more efficiently to membranes. Moreover, mutation of serine 36 to alanine significantly reduced membrane translocation of PKC Apl II. We suggest that translocation of nPKCs is regulated by phosphorylation of the C2 domain.     

A prostate-derived cDNA that is mapped to human chromosome 19 encodes a novel protein

The epithelial cells of prostate gland secrete various secretory products that play an important role in the growth and differentiation of prostate gland. These secretory products have also been implicated in neuroendocrine differentiation of benign prostatic hyperplasia and prostate malignancy. We have cloned a prostate-derived cDNA encoding a novel protein with a predicted molecular weight of 78 kDa (P(78)), and precisely mapped the cDNA sequence to chromosome 19. The P(78) gene has a complex genomic structure with 18 exons and 17 introns. The P(78) contains two conserved structural domains with limited similarity to domain D of synapsin I. The P(78) mRNA was expressed in various human cell lines. Western blot analysis using antibody specific for the P(78) revealed the presence of the P(78) protein in the prostate cancer cell lines with much lower level in metastatic prostate cancer cell lines compared to that in a primary prostate cancer cell line.     

A novel growth-inducible gene that encodes a protein with a conserved cold-shock domain.

We have isolated a cDNA that encodes a novel member of the Y-box binding protein family, termed as RYB-a (Rat Y-box Binding protein-a). RYB-a is a 31 kDa protein that contains a conserved cold-shock domain and an amino acid alignment similar to those of charge zipper proteins. Expression of RYB-a mRNA was highly abundant in the skeletal muscle, spleen, and fetal liver. The expression is very low in new-born and adult livers, suggesting its expression is under developmental regulation. In addition, the expression of RYB-a mRNA was induced in the liver during regeneration and by stimulation of quiescent fibroblast cells with serum. Induction in the fibroblasts was inhibited by treating the cell with a specific tyrosine kinase inhibitor, genistein or by detachment of cell-adhesion. Since both treatments are known to inhibit G1 cells to enter S phase, RYB-a gene is thought to be a member of growth-inducible genes.     

Novel phosphorylation site markers of protein kinase C delta activation

Protein kinase C delta (PKCdelta) is a Ser/Thr kinase which regulates numerous cellular processes, including proliferation, differentiation, migration and apoptosis. Here, we demonstrate that PKCdelta undergoes in vitro autophosphorylation at three sites within its V3 region (S299, S302, S304), each of which is unique to this PKC isoform and evolutionarily conserved. We demonstrate that S299 and S304 can be phosphorylated in mammalian cells following phorbol ester stimulation and that S299-phosphorylated PKCdelta is localised to both the plasma and nuclear membranes. These data indicate that PKCdelta is phosphorylated upon activation and that phospho-S299 represents a useful marker of the activated enzyme.     

Human brain n-chimaerin cDNA encodes a novel phorbol ester receptor.

A protein recognizing apolipoproteins AI, AII and AIV was purified from cultured mouse adipose cells of the Ob17MT18 clonal line. Apolipoprotein A binding sites were solubilized in the presence of proteinase inhibitors using the non-denaturating detergent CHAPS. Chromatography of the soluble extract on DEAE-Trisacryl was followed by immunoaffinity chromatography of the complex apolipoprotein AI-binding proteins on anti-(apolipoprotein AI) coupled to Sepharose 4B and then by h.p.l.c. on an RP-Select B column. A 1400-fold purification over the starting crude homogenate was achieved. The purified material contained two proteins that were both able to bind apolipoproteins AI, AII and AIV, but not low-density lipoprotein. Glycopeptidase F treatment showed the existence of a single protein bearing either N-linked high-mannose or complex oligosaccharide chains. The purified material showed an apparent molecular mass of 80 +/- 9 kDa by h.p.l.c. on a TSKG 3000 SW column. Rabbit polyclonal antibodies directed against the purified material revealed two protein bands of 80 and 92 kDa after SDS/PAGE under reducing conditions and immunoblotting. These bands were undetectable in growing Ob17PY cells previously shown not to bind the various apolipoproteins A and not to undergo cholesterol efflux, whereas they were conspicuous in growth-arrested Ob17PY cells which have recovered these properties.     

Mutation of a protein kinase C phosphorylation site in the erbB protein of avian erythroblastosis virus.

Tumor promoter-stimulated phosphorylation of threonine 98 of the erbB protein of avian erythroblastosis virus (AEV) correlates with inhibition of erbB-dependent mitogenesis. To more clearly define the role of phosphorylation of this residue in regulation of the activity of the erbB protein, we have constructed erbB mutations which encode alanine (Ala-98), tyrosine (Tyr-98), or serine (Ser-98) at position 98. The biosynthesis and stability of the three mutant proteins were similar to those of the wild-type erbB protein, and all three retained the ability to transform chicken embryo fibroblasts. Treatment of transformed CEF with 12-tetradecanoylphorbol-13-acetate (TPA) stimulated incorporation of 32Pi into wild-type and mutant erbB proteins and resulted in a slight decrease in the electrophoretic mobilities of all the erbB proteins. Tryptic maps of erbB phosphopeptides showed no endogenous or TPA-stimulated phosphorylation of alanine 98 or tyrosine 98 in cells transformed by the Ala-98 and Tyr-98 mutants. Analysis of tryptic phosphopeptides by high-pressure liquid chromatography revealed that TPA treatment of cells stimulated phosphorylation of other sites of the erbB protein in addition to threonine 98. A high endogenous level of phosphorylation of serine 98 of the Ser-98 mutant protein was found, and TPA treatment of cells did not result in further phosphorylation of this residue. Cells transformed by wild-type and mutant AEV were equally sensitive to TPA-dependent inhibition of growth in soft agar and TPA-dependent inhibition of [3H]thymidine incorporation. TPA treatment inhibited tyrosine phosphorylation to a similar extent in cells transformed by wild-type or Ala-98 AEV. These data indicate that phosphorylation of threonine 98 of the erbB protein is not responsible for TPA-dependent inhibition of growth of AEV-transformed cells or TPA-induced inhibition of erbB-dependent tyrosine phosphorylation. TPA-stimulated phosphorylation of the erbB protein at other sites may mediate these effects. The data also show that subtle changes in a phosphorylation site (i.e., changing threonine to serine) can drastically alter recognition by protein kinases.     

A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production

The C1 domain mediates the diacylglycerol (DAG)-dependent translocation of conventional and novel protein kinase C (PKC) isoforms. In novel PKC isoforms (nPKCs), this domain binds membranes with sufficiently high affinity to recruit nPKCs to membranes in the absence of any other targeting mechanism. In conventional PKC (cPKC) isoforms, however, the affinity of the C1 domain for DAG is two orders of magnitude lower, necessitating the coordinated binding of the C1 domain and a Ca2+-regulated C2 domain for translocation and activation. Here we identify a single residue that tunes the affinity of the C1b domain for DAG- (but not phorbol ester-) containing membranes. This residue is invariant as Tyr in the C1b domain of cPKCs and invariant as Trp in all other PKC C1 domains. Binding studies using model membranes, as well as live cell imaging studies of yellow fluorescent protein-tagged C1 domains, reveal that Trp versus Tyr toggles the C1 domain between a species with sufficiently high affinity to respond to agonist-produced DAG to one that is unable to respond to physiological levels of DAG. In addition, we show that while Tyr at this switch position causes cytosolic localization of the C1 domain under unstimulated conditions, Trp targets these domains to the Golgi, likely due to basal levels of DAG at this region. Thus, Trp versus Tyr at this key position in the C1 domain controls both the membrane affinity and localization of PKC. The finding that a single residue controls the affinity of the C1 domain for DAG-containing membranes provides a molecular explanation for why 1) DAG alone is sufficient to activate nPKCs but not cPKCs and 2) nPKCs target to the Golgi.     

A novel protein kinase C target site in protein kinase D is phosphorylated in response to signals for cardiac hypertrophy

Protein kinase D (PKD) regulates cardiac myocyte growth and contractility through phosphorylation of proteins such as class IIa histone deacetylases (HDACs) and troponin I (TnI). In response to agonists that activate G-protein-coupled receptors (GPCRs), PKD is phosphorylated by protein kinase C (PKC) on two serine residues (Ser-738 and Ser-742 in human PKD1) within an activation loop of the catalytic domain, resulting in stimulation of PKD activity. Here, we identify a novel PKC target site located adjacent to the auto-inhibitory pleckstrin homology (PH) domain in PKD. This site (Ser-412 in human PKD1) is conserved in each of the three PKD family members and is efficiently phosphorylated by multiple PKC isozymes in vitro. Employing a novel anti-phospho-Ser-412-specific antibody, we demonstrate that this site in PKD is rapidly phosphorylated in primary cardiac myocytes exposed to hypertrophic agonists, including norepinephrine (NE) and endothelin-1 (ET-1). Differential sensitivity of this event to pharmacological inhibitors of PKC, and data from in vitro enzymatic assays, suggest a predominant role for PKCδ in the control of PKD Ser-412 phosphorylation. Together, these data suggest a novel, signal-dependent mechanism for controlling PKD function in cardiac myocytes.