Phosphorylation of smooth muscle myosin light chain kinase by Ca2+/calmodulin-dependent protein kinase II: comparative study of the phosphorylation sites

Smooth muscle myosin light chain kinase (MLC-kinase) was rapidly phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) to a molar stoichiometry of 2.77 +/- 0.15 associated with a threefold increase in the concentration of calmodulin (CaM) required for half-maximal activation of MLC-kinase. Binding of CaM to MLC-kinase markedly reduced the phosphorylation stoichiometry to 0.21 +/- 0.05 and almost completely inhibited phosphorylation of sites in two peptides (32P-peptides P1 and P2) with reduced phosphorylation of peptide P3. By analogy, cAMP-dependent protein kinase phosphorylated MLC-kinase to a stoichiometry of 3.0 or greater in the absence of CaM with about a threefold decrease in the apparent affinity of MLC-kinase for CaM. Binding of CaM to MLC-kinase inhibited the phosphorylation to 0.84 +/- 0.13. Complete tryptic digests contained two major 32P-peptides as reported previously. One of the peptides, whose phosphorylation was inhibited in the presence of excess calmodulin, appeared to be the same as P2. Automated Edman sequence analysis suggested that both CaM-kinase II and cAMP-dependent protein kinase phosphorylated this peptide at the second of the two adjacent serine residues located at the C-terminal boundary of the CaM-binding domain. However, the other peptide phosphorylated by cAMP-dependent protein kinase, regardless of whether CaM was bound, was different from P1 and P3. Thus, MLC-kinase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of CaM-kinase II and is blocked by Ca2+/CaM-binding.     

Phosphorylation of smooth muscle caldesmon by calmodulin-dependent protein kinase II. Identification of the phosphorylation sites

Smooth muscle caldesmon was phosphorylated by smooth muscle calmodulin-dependent protein kinase II. The extent of phosphorylation obtained was 5.65 mol of phosphate/mol of caldesmon. Phosphorylated protein was subjected to the complete trypsin proteolysis and the produced phosphopeptides were purified by C-8 reverse phase chromatography. Nine phosphopeptides were isolated and by amino acid sequence analysis, eight phosphorylation sites were identified. According to the published amino acid sequence of chicken gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G., and Lin, W.-G. (1989) J. Biol. Chem. 264, 13873-13879), these sites were serine 26, serine 59, serine 73, threonine 469, serine 475, serine 587, serine 620, and serine 726. The time course of phosphorylation of these sites was also measured and it was concluded that the first site was serine 73, the second site was serine 26, the third site was serine 726, and the fourth site was serine 587. The preferred phosphorylation sites were located in the amino terminus myosin binding domain whereas slower phosphorylation occurred in the carboxyl terminus actin/calmodulin domain.     

Site-specific dephosphorylation of smooth muscle myosin light chain kinase by protein phosphatases 1 and 2A.

Smooth muscle myosin light chain kinase is phosphorylated at two sites (A and B) by different protein kinases. Phosphorylation at site A increases the concentration of Ca2+/calmodulin required for kinase activation. Diphosphorylated myosin light chain kinase was used to determine the site-specificity of several forms of protein serine/threonine phosphatase. These phosphatases readily dephosphorylated myosin light chain kinase in vitro and displayed differing specificities for the two phosphorylation sites. Type 2A protein phosphatase specifically dephosphorylated site A, and binding of Ca2+/calmodulin to the kinase had no effect on dephosphorylation. The purified catalytic subunit of type 1 protein phosphatase dephosphorylated both sites in the absence of Ca2+/calmodulin but only dephosphorylated site A in the presence of Ca2+/calmodulin. A protein phosphatase fraction was prepared from smooth muscle actomyosin by extraction with 80 mM MgCl2. On the basis of sensitivity to okadaic acid and inhibitor 2, this activity was composed of multiple protein phosphatases including type 1 activity. This phosphatase fraction dephosphorylated both sites in the absence of Ca2+/calmodulin. However, dephosphorylation of both sites A and B was completely blocked in the presence of Ca2+/calmodulin. These results indicate that two phosphorylation sites of myosin light chain kinase are dephosphorylated by multiple protein serine/threonine phosphatases with unique catalytic specificities.     

Myosin light chain kinase phosphorylation in tracheal smooth muscle

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+.     

Ca2+/calmodulin-dependent kinase II phosphorylates the epidermal growth factor receptor on multiple sites in the cytoplasmic tail and serine 744 within the kinase domain to regulate signal generation.

Down-regulation of receptor tyrosine kinase activity plays an essential role in coordinating and controlling cellular growth/differentiation. Ca2+/calmodulin-dependent kinase II (CaM kinase II)-mediated phosphorylation of threonine 1172 in the cytoplasmic tail of HER2/c-erbB2 can modulate tyrosine kinase activity and consensus phosphorylation sites are also found at serines 1046/1047 in the structurally related epidermal growth factor receptor (EGFR). We show that serines 1046/1047 are sites for CaM kinase II phosphorylation, although there is a preference for serine 1047, which resides within the consensus -R-X-X-S-. In addition, we have identified major phosphorylation sites at serine 1142 and serine 1057, which lie within a novel -S-X-D- consensus. Mutation of serines 1046/1047 in full-length EGFR enhanced both fibroblast transformation and tyrosine autokinase activity that was significantly potentiated by additional mutation of serines 1057 and 1142. A single CaM kinase II site was also identified at serine 744 within sub-kinase domain III, and autokinase activity was significantly affected by mutation of this serine to an aspartic acid making this site appear constitutively phosphorylated. We have addressed the mechanism by which CaM kinase II phosphorylation of the EGFR might regulate receptor autokinase activity and show that this modification can hinder association of the cytoplasmic tail with the kinase domain to prevent an enzyme-substrate interaction. We postulate that the location and greater number of CaM kinase II phosphorylation sites in the EGFR compared with HER-2/c-erbB2, leading to differential regulation of autokinase activity, contributes to differences in the strength of downstream signaling events and may explain the higher relative transforming potential of HER-2/cerbB2.     

Regulation of calcineurin by phosphorylation. Identification of the regulatory site phosphorylated by Ca2+/calmodulin-dependent protein kinase II and protein kinase C

The site in calcineurin, the Ca2+/calmodulin (CaM)-dependent protein phosphatase, which is phosphorylated by Ca2+/CaM-dependent protein kinase II (CaM-kinase II) has been identified. Analyses of 32P release from tryptic and cyanogen bromide peptides derived from [32P]calcineurin plus direct sequence determination established the site as -Arg-Val-Phe-Ser(PO4)-Val-Leu-Arg-, which conformed to the consensus phosphorylation sequence for CaM-kinase II (Arg-X-X-Ser/Thr-). This phosphorylation site is located at the C-terminal boundary of the putative CaM-binding domain in calcinerin (Kincaid, R. L., Nightingale, M. S., and Martin, B. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8983-8987), thereby accounting for the observed inhibition of this phosphorylation when Ca2+/CaM is bound to calcineurin. Since the phosphorylation site sequence also contains elements of the specificity determinants for Ca2+/phospholipid-dependent protein kinase (protein kinase C) (basic residues both N-terminal and C-terminal to Ser/Thr), we tested calcineurin as a substrate for protein kinase C. Protein kinase C catalyzed rapid stoichiometric phosphorylation, and the characteristics of the reaction were the same as with CaM-kinase II: 1) the phosphorylation was blocked by binding of Ca2+/CaM to calcineurin; 2) phosphorylation partially inactivated calcineurin by increasing the Km (from 9.9 +/- 1.1 to 17.5 +/- 1.1 microM 32P-labeled myosin light chain); and 3) [32P]calcineurin exhibited very slow autodephosphorylation but was rapidly dephosphorylated by protein phosphatase IIA. Tryptic and thermolytic 32P-peptide mapping and sequential phosphoamino acid sequence analysis confirmed that protein kinase C and CaM-kinase II phosphorylated the same site.     

Phosphorylation of calcineurin: effect on calmodulin binding

The effect of phosphorylation of calcineurin on calmodulin (CaM) binding was examined using a synthetic peptide which contains the CaM-binding domain and the serine phosphorylation site. The peptide, corresponding to residues 391-414 of brain calcineurin A subunit, was rapidly phosphorylated by protein kinase C and Ca2+/CaM-dependent protein kinase II but not by cAMP-dependent protein kinase. Phosphorylation of peptide 391-414 did not significantly alter the binding of CaM when compared to the non-phosphorylated peptide.     

Phosphorylation of smooth muscle myosin light chain kinase by protein kinase C. Comparative study of the phosphorylated sites

Smooth muscle myosin light chain kinase is phosphorylated in vitro by protein kinase C purified from human platelets. When myosin light chain kinase which has calmodulin bound is phosphorylated by protein kinase C, 0.8-1.1 mol of phosphate is incorporated per mol of myosin light chain kinase with no effect on its enzyme activity. Phosphorylation of myosin light chain kinase with no calmodulin bound results in the incorporation of 2-2.4 mol of phosphate and significantly decreases the rate of myosin light chain kinase activity. The decrease in myosin light chain kinase activity is due to a 3.3-fold increase in the concentration of calmodulin necessary for the half-maximal activation of myosin light chain kinase. The sites phosphorylated by protein kinase C and the catalytic subunit of cAMP-dependent protein kinase were compared by two-dimensional peptide mapping following extensive tryptic digestion of phosphorylated myosin light chain kinase. The single site phosphorylated by protein kinase C when calmodulin is bound to myosin light chain kinase (site 3) is different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 1). The additional site that is phosphorylated by protein kinase C when calmodulin is not bound appears to be the same site phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 2). These studies confirm the important role of site 2 in binding calmodulin to myosin light chain kinase. Sequential studies using both protein kinase C and the catalytic subunit of cAMP-dependent protein kinase suggest that the phosphorylation of site 1 also plays a part in decreasing the affinity of myosin light chain kinase for calmodulin.     

Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase

Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver. The calmodulin (CaM)-dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM-dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP-dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20-30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes.     

Phosphorylation of microtubule-associated protein tau by Ca2+/calmodulin-dependent protein kinase II in its tubulin binding sites

The paired helical filaments (PHF) found in Alzheimer's disease (AD) brain are composed mainly of the hyperphosphorylated form of microtubule-associated protein tau (PHF-tau). It is well known that tau is a good in vitro substrate for Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). To establish the phosphorylation sites, the longest human tau (hTau40) was bacterially expressed and phosphorylated by CaM kinase II, followed by digestion with lysyl endoprotease. The digests were subjected to liquid chromatography/mass spectrometry. We found that 5 of 22 identified peptides were phosphorylated. From the tandem mass spectrometry, two phosphorylation sites (serines 262 and 356) were identified in the tubulin binding sites. When tau was phosphorylated by CaM kinase II, the binding of tau to taxol-stabilized microtubules was remarkably impaired. As both serines 262 and 356 are reportedly phosphorylated in PHF-tau, CaM kinase II may be involved in hyperphosphorylation of tau in AD brain.     

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)     

Selective binding of L-thyroxine by myosin light chain kinase

L-Thyroxine selectively inhibited Ca2+-calmodulin-activated myosin light chain kinases (MLC kinase) purified from rabbit skeletal muscle, chicken gizzard smooth muscle, bovine thyroid gland, and human platelet with similar Ki values (Ki = 2.5 microM). A detailed analysis of L-thyroxine inhibition of smooth muscle myosin light chain kinase activation was undertaken in order to determine the effect of L-thyroxine on the stoichiometries of Ca2+, calmodulin, and the enzyme in the activation process. The kinetic data indicated that L-thyroxine does not interact with calmodulin but, instead, through direct association with the enzyme, inhibits the binding of the Ca2+-calmodulin complex to MLC kinase. L-[125I]Thyroxine gel overlay revealed that the 95-kDa fragment of chicken gizzard MLC kinase digested by chymotrypsin and all the fragments of 110, 94, 70, and 43 kDa produced by Staphylococcus aureus V8 protease digestion which contain the calmodulin binding domain retain L-[125I]thyroxine binding activity, whereas smaller peptides were not radioactive. Since MLC kinase is phosphorylated by cAMP-dependent protein kinase (2 mol of phosphate/mol of MLC kinase), the effect of L-thyroxine on the phosphorylation of MLC kinase also was examined. L-Thyroxine binding did not inhibit the phosphorylation of MLC kinase and, moreover, reversed the inhibition of phosphorylation obtained with the calmodulin-enzyme complex. These observations support the suggestion that L-thyroxine binds at or near the calmodulin-binding site of MLC kinase. L-Thyroxine may serve as a different type of pharmacological tool for elucidating the biological significance of MLC kinase-mediated reactions.     

AMP-activated protein kinase phosphorylates and desensitizes smooth muscle myosin light chain kinase

Smooth muscle contraction is initiated by a rise in intracellular calcium, leading to activation of smooth muscle myosin light chain kinase (MLCK) via calcium/calmodulin (CaM). Activated MLCK then phosphorylates the regulatory myosin light chains, triggering cross-bridge cycling and contraction. Here, we show that MLCK is a substrate of AMP-activated protein kinase (AMPK). The phosphorylation site in chicken MLCK was identified by mass spectrometry to be located in the CaM-binding domain at Ser(815). Phosphorylation by AMPK desensitized MLCK by increasing the concentration of CaM required for half-maximal activation. In primary cultures of rat aortic smooth muscle cells, vasoconstrictors activated AMPK in a calcium-dependent manner via CaM-dependent protein kinase kinase-beta, a known upstream kinase of AMPK. Indeed, vasoconstrictor-induced AMPK activation was abrogated by the STO-609 CaM-dependent protein kinase kinase-beta inhibitor. Myosin light chain phosphorylation was increased under these conditions, suggesting that contraction would be potentiated by ablation of AMPK. Indeed, in aortic rings from mice in which alpha1, the major catalytic subunit isoform in arterial smooth muscle, had been deleted, KCl- or phenylephrine-induced contraction was increased. The findings suggest that AMPK attenuates contraction by phosphorylating and inactivating MLCK. This might contribute to reduced ATP turnover in the tonic phase of smooth muscle contraction.     

Phosphorylation of tau at serine 416 by Ca2+/calmodulin-dependent protein kinase II in neuronal soma in brain

It is well known that tau is a good in vitro substrate for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). However, it is not clear at present whether CaM kinase II phosphorylates tau in vivo or not. Serine 416, numbered according to the longest human tau isoform, has been reported to be one of the major phosphorylation sites by CaM kinase II in vitro. In this study, we produced a specific antibody against tau phosphorylated at serine 416 (PS416-tau). Immunoblot analysis revealed that the antibody reacted with tau in the rat brain extract which was prepared in the presence of protein phosphatase inhibitors. Developmental study indicated that serine 416 was strongly phosphorylated at early developmental stages in rat brain. We examined the localization of PS416-tau in primary cultured hippocampal neurons and the immortalized GnRH neurons (GT1-7 cells), which were stably transfected with CaM kinase IIalpha cDNA. Immunostaining of these cells indicated that tau was phosphorylated mainly in neuronal soma. Interestingly, tau in neuronal soma in Alzheimer's disease (AD) brain was strongly immunostained by the antibody. These results suggest that CaM kinase II is involved in the accumulation of tau in neuronal soma in AD brain.     

A calmodulin-dependent protein kinase from lower eukaryote Physarum polycephalum

A full-length cDNA coding a calmodulin (CaM)-dependent protein kinase gene was cloned from Physarum plasmodia poly(A)-RNA by polymerase chain reaction with the oligonucleotide primers that were designed after the amino acid sequence of highly conserved regions of myosin light-chain kinase. Sequence analysis of the cDNA revealed that this Physarum kinase was a 42,519-Da protein with an ATP-binding domain, Ser/Thr kinase active site signature, and CaM-binding domain. Expression of the cDNA in Escherichia coli demonstrated that the Physarum kinase in the presence of Ca2+ and CaM phosphorylated the recombinant phosphorylatable light chain (PLc) of Physarum myosin II. The peptide analysis after proteolysis of the phosphorylated PLc indicated that Ser 18 was phosphorylated. The site was confirmed by the failure of phosphorylation of PLc, the Ser 18 of which was replaced by Ala. The physiological role of the kinase will be discussed with special reference to the 55-kDa kinase, which had been previously purified from Physarum plasmodia for phosphorylated PLc.     

Phosphorylation of smooth muscle myosin light chain kinase by Ca2+-activated, phospholipid-dependent protein kinase

Protein kinase C incorporates phosphate into two sites of myosin light chain kinase (MLC-kinase) in the absence of calmodulin. Phosphorylation is all but abolished in the presence of Ca2+ and calmodulin, suggesting that both sites of phosphorylation are close to the calmodulin binding site. The phosphorylation of MLC-kinase results in an approximately 10-fold increase in the dissociation constant of MLC-kinase for calmodulin. Following phosphorylation (2 mol/mol of enzyme) of MLC-kinase by protein kinase C, an additional 2 mol of phosphate can be incorporated into the MLC-kinase apoenzyme by the cAMP-dependent protein kinase. Different maps of phosphopeptides were obtained by tryptic hydrolysis from MLC-kinase preparations phosphorylated by each kinase. The phosphorylation sites for the cAMP-dependent kinase were located in a fragment of approximately 25,000 daltons. In contrast the phosphorylation sites for protein kinase C are found in a much smaller tryptic peptide. These results suggest that the phosphorylation sites on MLC-kinase are different for protein kinase C and for cAMP-dependent protein kinase. However, phosphorylation in both regions results in a reduced affinity for calmodulin.     

Phosphorylation of calmodulin by Ca2+/calmodulin-dependent protein kinase IV

Calmodulin-dependent protein kinase IV (CaM-kinase IV) phosphorylated calmodulin (CaM), which is its own activator, in a poly-L-Lys [poly(Lys)]-dependent manner. Although CaM-kinase II weakly phosphorylated CaM under the same conditions, CaM-kinase I, CaM-kinase kinase alpha, and cAMP-dependent protein kinase did not phosphorylate CaM. Polycations such as poly(Lys) were required for the phosphorylation. The optimum concentration of poly(Lys) for the phosphorylation of 1 microM CaM was about 10 microg/ml, but poly(Lys) strongly inhibited CaM-kinase IV activity toward syntide-2 at this concentration, suggesting that the phosphorylation of CaM is not due to simple activation of the catalytic activity. Poly-L-Arg could partially substitute for poly(Lys), but protamine, spermine, and poly-L-Glu/Lys/Tyr (6/3/1) could not. When phosphorylation was carried out in the presence of poly(Lys) having various molecular weights, poly(Lys) with a higher molecular weight resulted in a higher degree of phosphorylation. Binding experiments using fluorescence polarization suggested that poly(Lys) mediates interaction between the CaM-kinase IV/CaM complex and another CaM. The 32P-labeled CaM was digested with BrCN and Achromobacter protease I, and the resulting peptides were purified by reversed-phase HPLC. Automated Edman sequence analysis of the peptides, together with phosphoamino acid analysis, indicated that the major phosphorylation site was Thr44. Activation of CaM-kinase II by the phosphorylated CaM was significantly lower than that by the nonphosphorylated CaM. Thus, CaM-kinase IV activated by binding Ca2+/CaM can bind and phosphorylate another CaM with the aid of poly(Lys), leading to a decrease in the activity of CaM.     

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.     

Phosphorylation of neuromodulin (GAP-43) by casein kinase II. Identification of phosphorylation sites and regulation by calmodulin.

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin-binding protein believed to play a role in regulation of neurite outgrowth and neuroplasticity. Neuromodulin is phosphorylated by protein kinase C, and this phosphorylation prevents calmodulin from binding to neuromodulin (Alexander, K. A., Cimler, B. M., Meier, K. E. & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). The only other protein kinase known to phosphorylate neuromodulin is casein kinase II (Pisano, M. R., Hegazy, M. G., Reimann, E. M. & Dokas, L. A. (1988) Biochem. Biophys. Res. Commun. 155, 1207-1212). Phosphoamino acid analyses revealed that casein kinase II modified serine and threonine residues in both native bovine and recombinant mouse neuromodulin. Two serines located in the C-terminal end of neuromodulin, Ser-192 and Ser-193, were identified as the major casein kinase II phosphorylation sites. Thr-88, Thr-89, or Thr-95 were identified as minor casein kinase II phosphorylation sites. Phosphorylation by casein kinase II did not affect the ability of neuromodulin to bind to calmodulin-Sepharose. However, calmodulin did inhibit the phosphorylation of neuromodulin by casein kinase II with a Ki of 1-2 microM. Calmodulin inhibition of casein kinase II phosphorylation was due to calmodulin binding to neuromodulin rather than to the protein kinase. These data suggest that the minimal secondary and tertiary structure exhibited by neuromodulin may be sufficient to juxtapose its calmodulin-binding domain, located at the N-terminal end, with the neuromodulin casein kinase II phosphorylation sites at the C-terminal end of the protein. We propose that calmodulin regulates casein kinase II phosphorylation of neuromodulin by binding to neuromodulin and sterically hindering the interaction of casein kinase II with its phosphorylation sites on neuromodulin.     

The regulation of death-associated protein (DAP) kinase in apoptosis

DAP-kinase is a calcium/calmodulin (Ca2+/CaM) serine/threonine kinase which positively mediates programmed cell death in a variety of cell systems. The kinase is localized to the actin microfilament and has a unique, multidomain structure consisting of ankyrin repeats and a death domain. One of the substrates of DAP-kinase was identified as myosin light chain (MLC), the phosphorylation of which mediates membrane blebbing. Another arm in its mode of action leads to the formation of autophagic vesicles. Recent work addressed its mode of regulation and identified a mechanism which restrains its apoptotic function in growing cells and enables its activation during cell death. It involves an inhibitory type of autophosphorylation on serine 308 within the CaM regulatory domain. This negative phosphorylation takes place in growing cells and is strongly reduced upon their exposure to the apoptotic stimulus of C6-ceramide. The substitution of serine 308 to alanine, which mimics the ceramide-induced dephosphorylation at this site, increases Ca2+/CaM-independent substrate phosphorylation, as well as binding and overall sensitivity of the kinase to CaM. At the cellular level, it strongly enhances the death-promoting activity of the kinase. These results are consistent with a molecular model in which phosphorylation on serine 308 stabilizes a locked conformation of the CaM regulatory domain within the catalytic cleft and, simultaneously, also interferes with CaM binding. We propose that this unique mechanism of auto-inhibition evolved to impose a locking device which keeps DAP-kinase silent in healthy cells and ensures its activation only in response to apoptotic signals.