Regulatory mechanism of Dictyostelium myosin light chain kinase A

In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin-dependent protein kinase kinase as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr166 by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an approximately 140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by activated MLCK-A. Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr289 and that, subsequent to Thr166 phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Ser residues. Using site-directed mutagenesis, we show that autophosphorylation at Thr289 is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283-288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr166 phosphorylation. Simple removal of this region resulted in a significant increase in the kcat of MLCK-A; however, it did not generate maximum enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr166 phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme.     

Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation

Autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase converts it from a Ca2(+)-dependent to a Ca2(+)-independent or autonomous kinase, a process that may underlie some long-term enhancement of transient Ca2+ signals. We demonstrate that the neuronal alpha subunit clone expressed in COS-7 cells (alpha-CaM kinase) is sufficient to encode the regulatory phenomena characteristic of the multisubunit kinase isolated from brain. Activity of alpha-CaM kinase is highly dependent on Ca2+/calmodulin. It is converted by autophosphorylation to an enzyme capable of Ca2(+)-independent (autonomous) substrate phosphorylation and autophosphorylation. Using site-directed mutagenesis, we separately eliminate five putative autophosphorylation sites within the regulatory domain and directly examine their individual roles. Ca2+/calmodulin-dependent kinase activity is fully retained by each mutant, but Thr286 is unique among the sites in being indispensable for generation of an autonomous kinase.     

Activation of type II calcium/calmodulin-dependent protein kinase by Ca2+/calmodulin is inhibited by autophosphorylation of threonine within the calmodulin-binding domain

It is now well established that autophosphorylation of a threonine residue located next to each calmodulin-binding domain in the subunits of type II Ca2+/calmodulin-dependent protein kinase causes the kinase to remain active, although at a reduced rate, after Ca2+ is removed from the reaction. This autophosphorylated form of the kinase is still sensitive to Ca2+/calmodulin, which is required for a maximum catalytic rate. After removal of Ca2+, new sites are autophosphorylated by the partially active kinase. Autophosphorylation of these sites abolishes sensitivity of the kinase to Ca2+/calmodulin (Hashimoto, Y., Schworer, C. M., Colbran, R. J., and Soderling, T. R. (1987) J. Biol. Chem. 262, 8051-8055). We have identified two pairs of homologous residues, Thr305 and Ser314 in the alpha subunit and Thr306 and Ser315 in the beta subunit, that are autophosphorylated only after removal of Ca2+ from an autophosphorylation reaction. The sites were identified by direct sequencing of labeled tryptic phosphopeptides isolated by reverse-phase high pressure liquid chromatography. Thr305-306 is rapidly dephosphorylated by purified protein phosphatases 1 and 2A, whereas Ser314-315 is resistant to dephosphorylation. We have shown by selective dephosphorylation that the presence of phosphate on Thr305-306 blocks sensitivity of the kinase to Ca2+/calmodulin. In contrast, the presence of phosphate on Ser314-315 is associated with an increase in the Kact for Ca2+/calmodulin of only about 2-fold, producing a relatively small decrease in sensitivity to Ca2+/calmodulin.     

Phosphorylation of smooth muscle heavy meromyosin by calcium-activated, phospholipid-dependent protein kinase. The effect on actin-activated MgATPase activity

Smooth muscle heavy meromyosin (HMM) can serve as a substrate for the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) as well as for the Ca2+/calmodulin-dependent kinase, myosin light chain kinase. When turkey gizzard HMM is incubated with protein kinase C, 1.7-2.2 mol of phosphate are incorporated per mol of HMM, all of it into the 20,000-Da light chain of HMM. Two-dimensional peptide mapping following tryptic hydrolysis revealed that protein kinase C phosphorylated a different site on the 20,000-Da HMM light chain than did myosin light chain kinase. Moreover, sequential phosphorylation of HMM by myosin light chain kinase and protein kinase C resulted in the incorporation of 4 mol of phosphate/mol of HMM, i.e. 2 mol of phosphate into each 20,000-Da light chain. When unphosphorylated HMM was phosphorylated by myosin light chain kinase, its actin-activated MgATPase activity increased from 4 nmol to 156 nmol of phosphate released/mg of HMM/min. Subsequent phosphorylation of this phosphorylated HMM by protein kinase C decreased the actin-activated MgATPase activity of HMM to 75 nmol of phosphate released/mg of HMM/min.     

Massive autophosphorylation of the Ser/Thr-rich domain controls protein kinase activity of TRPM6 and TRPM7

TRPM6 and TRPM7 are bifunctional proteins expressing a TRP channel fused to an atypical alpha-kinase domain. While the gating properties of TRPM6 and TRPM7 channels have been studied in detail, little is known about the mechanisms regulating kinase activity. Recently, we found that TRPM7 associates with its substrate myosin II via a kinase-dependent mechanism suggesting a role for autophosphorylation in substrate recognition. Here, we demonstrate that the cytosolic C-terminus of TRPM7 undergoes massive autophosphorylation (32+/-4 mol/mol), which strongly increases the rate of substrate phosphorylation. Phosphomapping by mass spectrometry indicates that the majority of autophosphorylation sites (37 out of 46) map to a Ser/Thr-rich region immediately N-terminal of the catalytic domain. Deletion of this region prevents substrate phosphorylation without affecting intrinsic catalytic activity suggesting that the Ser/Thr-rich domain contributes to substrate recognition. Surprisingly, the TRPM6-kinase is regulated by an analogous mechanism despite a lack of sequence conservation with the TRPM7 Ser/Thr-rich domain. In conclusion, our findings support a model where massive autophosphorylation outside the catalytic domain of TRPM6 and TRPM7 may facilitate kinase-substrate interactions leading to enhanced phosphorylation of those substrates.     

Autophosphorylation of smooth-muscle caldesmon.

Caldesmon, a major actin- and calmodulin-binding protein of smooth muscle, has been implicated in regulation of the contractile state of smooth muscle. The isolated protein can be phosphorylated by a co-purifying Ca2+/calmodulin-dependent protein kinase, and phosphorylation blocks inhibition of the actomyosin ATPase by caldesmon [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have examined the phosphorylation of caldesmon in more detail. Several lines of evidence indicate that caldesmon itself is a kinase and the reaction is an intermolecular autophosphorylation: (1) caldesmon (141 kDa) and a 93 kDa proteolytic fragment of caldesmon can be separated by ion-exchange chromatography: both retain caldesmon kinase activity, which is Ca2+/calmodulin-dependent; (2) chymotryptic digestion of caldesmon generates a Ca2+/calmodulin-independent form of caldesmon kinase; (3) caldesmon purified to electrophoretic homogeneity retains caldesmon kinase activity, and elution of enzymic activity from a fast-performance-liquid-chromatography ion-exchange column correlates with caldesmon of Mr 141,000; (4) caldesmon is photoaffinity-labelled with 8-azido-[alpha-32P]ATP; labelling is inhibited by ATP, GTP and CTP, indicating a lack of nucleotide specificity; (5) caldesmon binds tightly to Affi-Gel Blue resin, which recognizes proteins having a dinucleotide fold. Autophosphorylation of caldesmon occurs predominantly on serine residues (83.3%), with some threonine (16.7%) and no tyrosine phosphorylation. Autophosphorylation is site-specific: 98% of the phosphate incorporated is recovered in a 26 kDa chymotryptic peptide. Complete tryptic/chymotryptic digestion of this phosphopeptide followed by h.p.l.c. indicates three major phosphorylation sites. Caldesmon exhibits a high degree of substrate specificity: apart from autophosphorylation, brain synapsin I is the only good substrate among many potential substrates examined. These observations indicate that caldesmon may regulate its own function (inhibition of the actomyosin ATPase) by Ca2+/calmodulin-dependent autophosphorylation. Furthermore, caldesmon may regulate other cellular processes, e.g. neurotransmitter release, through the Ca2+/calmodulin-dependent phosphorylation of other proteins such as synapsin I.     

Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase

The phosphorylation of the calmodulin-dependent enzyme myosin light chain kinase, purified from bovine tracheal smooth muscle and human blood platelets, by the catalytic subunit of cAMP-dependent protein kinase and by cGMP-dependent protein kinase was investigated. When myosin light chain kinase which has calmodulin bound is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of tracheal myosin light chain kinase or platelet myosin light chain kinase, with no effect on the catalytic activity. Phosphorylation when calmodulin is not bound results in the incorporation of 2 mol of phosphate and significantly decreases the activity. The decrease in myosin light chain kinase activity is due to a 5 to 7-fold increase in the amount of calmodulin required for half-maximal activation of both tracheal and platelet myosin light chain kinase. In contrast to the results with the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase cannot phosphorylate tracheal myosin light chain kinase in the presence of bound calmodulin. When calmodulin is not bound to tracheal myosin light chain kinase, cGMP-dependent protein kinase phosphorylates only one site, and this phosphorylation has no effect on myosin light chain kinase activity. On the other hand, cGMP-dependent protein kinase incorporates phosphate into two sites in platelet myosin light chain kinase when calmodulin is not bound. The sites phosphorylated by the two cyclic nucleotide-dependent protein kinases were compared by two-dimensional peptide mapping following extensive tryptic digestion of the phosphorylated myosin light chain kinases. With respect to the tracheal myosin light chain kinase, the single site phosphorylated by cGMP-dependent protein kinase when calmodulin is not bound appears to be the same site phosphorylated in the tracheal enzyme by the catalytic subunit of cAMP-dependent protein kinase when calmodulin is bound. With respect to the platelet myosin light chain kinase, the additional site that was phosphorylated by cGMP-dependent protein kinase when calmodulin was not bound was different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.     

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+.     

Inhibitory autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase analyzed by site-directed mutagenesis.

Initial autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) occurs at Thr286 (the "autonomy" site) and converts the kinase from a Ca(2+)-dependent to a partially Ca(2+)-independent or autonomous enzyme. After removal of Ca2+/calmodulin, the autonomous kinase undergoes a "burst" of inhibitory autophosphorylation at sites distinct from the autonomy site which may be masked in the presence of bound calmodulin. This burst of Ca(2+)-independent autophosphorylation blocks the ability of calmodulin to activate the kinase. We have used site-directed mutagenesis to replace putative inhibitory autophosphorylation sites within the calmodulin binding domain of recombinant alpha-CaM kinase with nonphosphorylatable alanines and examined the effects on autophosphorylation, kinase activity, and calmodulin binding. Although prominent Ca(2+)-independent autophosphorylation occurs within the calmodulin binding domain at Thr305, Thr306, and Ser314 in wild-type alpha-CaM kinase, the inhibitory effect on kinase activity and calmodulin binding is retained in mutants lacking any one of these three sites. However, when both Thr305 and Thr306 are converted to alanines the kinase does not display inhibition of either activity or calmodulin binding. Autophosphorylation at either Thr305 or Thr306 is therefore sufficient to block both binding and activation of the kinase by Ca2+/calmodulin. Thr306 is also slowly autophosphorylated in a basal reaction in the continuous absence of Ca2+/calmodulin. Autophosphorylation of Thr306 by the kinase in either its basal or autonomous state suggests that in the absence of bound calmodulin, the region of the autoregulatory domain surrounding Thr306, rather than the region near the autonomy site, lies nearest the peptide substrate binding site of the kinase.     

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.     

Regulation of embryonic smooth muscle myosin by protein kinase C

Phosphorylation of the 20-kDa light chain regulates adult smooth muscle myosin; phosphorylation by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase stimulates the actomyosin ATPase activity of adult smooth muscle myosin; the simultaneous phosphorylation of a separate site on the 20-kDa light chain by the Ca2+/phospholipid-dependent enzyme protein kinase C attenuates the myosin light chain kinase-induced increase in the actomyosin ATPase activity of adult myosin. Fetal smooth muscle myosin, purified from 12-day-old fertilized chicken eggs, is structurally different from adult smooth muscle myosin. Nevertheless, phosphorylation of a single site on the 20-kDa light chain of fetal myosin by myosin light chain kinase results in stimulation of the actomyosin ATPase activity of this myosin. Protein kinase C, in contrast, phosphorylates three sites on the fetal myosin 20-kDa light chain including a serine or threonine residue on the same peptide phosphorylated by myosin light chain kinase. Interestingly, phosphorylation by protein kinase C stimulates the actomyosin ATPase activity of fetal myosin. Moreover, unlike adult myosin, there is no attenuation of the actomyosin ATPase activity when fetal myosin is simultaneously phosphorylated by myosin light chain kinase and protein kinase C. These data demonstrate, for the first time, the in vitro activation of a smooth muscle myosin by another enzyme besides myosin light chain kinase and raise the possibility of alternate pathways for regulating smooth muscle myosin in vivo.     

Phosphorylation by protein kinase C of the 20,000-dalton light chain of myosin in intact and chemically skinned vascular smooth muscle

In the present study we tested the hypothesis that phosphorylation of the 20,000-dalton light chain subunit of smooth muscle myosin (LC20) by the calcium-activated and phospholipid-dependent protein kinase C regulates contraction of chemically-permeabilized (glycerinated) porcine carotid artery smooth muscle. Purified protein kinase C and oleic acid were used to phosphorylate LC20 in glycerinated muscles in the presence of a CaEGTA/EGTA buffer system (pCa 8) to prevent activation of myosin light chain kinase. Phosphorylation of the light chain to 1.3 mol of PO4/mol of LC20 did not stimulate contraction. Tryptic digests of glycerinated carotid artery LC20 contained two major phosphopeptides which contained phosphoserine but not phosphothreonine. Incubation of glycerinated muscles with calcium (20 microM) and calmodulin (10 microM) resulted in contraction and LC20 phosphorylation to 1.1 mol of PO4/mol of LC20; tryptic digests of LC20 from these muscles contained a single phosphopeptide which could be distinguished by phosphopeptide mapping from the two phosphopeptides derived from muscles phosphorylated with protein kinase C. Further phosphorylation of Ca2+/calmodulin-activated muscles to 2.0 mol of PO4/mol of LC20, by incubation with protein kinase C, had no effect on either the level of isometric force or the lightly-loaded shortening velocity (after-load = 0.1 peak active force); removal of Ca2+ and calmodulin, but not protein kinase C and oleic acid, resulted in normal relaxation in spite of maintained phosphorylation to 1.2 mol of PO4/mol of LC20. Comparison of LC20 phosphopeptide maps from glycerinated muscles incubated with protein kinase C plus Ca2+/calmodulin (2.0 mol of PO4/mol of LC20) to maps from intact muscles stimulated with 10(-6) M phorbol 12,13-dibutyrate (0.05 mol of PO4/mol of LC20) showed that the same three phosphopeptides were present in both the intact and glycerinated muscles. These findings show that phosphorylation of LC20 by protein kinase C in glycerinated muscles to levels at least 40 times higher than those present during contraction of intact, phorbol ester-stimulated muscles does not activate contraction nor does it significantly modify the contraction of smooth muscle which occurs in response to the Ca2+/calmodulin-dependent phosphorylation of Ser19 by myosin light chain kinase.     

Myosin regulatory light chain diphosphorylation slows relaxation of arterial smooth muscle

The principal signal to activate smooth muscle contraction is phosphorylation of the regulatory light chains of myosin (LC(20)) at Ser(19) by Ca(2+)/calmodulin-dependent myosin light chain kinase. Inhibition of myosin light chain phosphatase leads to Ca(2+)-independent phosphorylation at both Ser(19) and Thr(18) by integrin-linked kinase and/or zipper-interacting protein kinase. The functional effects of phosphorylation at Thr(18) on steady-state isometric force and relaxation rate were investigated in Triton-skinned rat caudal arterial smooth muscle strips. Sequential phosphorylation at Ser(19) and Thr(18) was achieved by treatment with adenosine 5'-O-(3-thiotriphosphate) in the presence of Ca(2+), which induced stoichiometric thiophosphorylation at Ser(19), followed by microcystin (phosphatase inhibitor) in the absence of Ca(2+), which induced phosphorylation at Thr(18). Phosphorylation at Thr(18) had no effect on steady-state force induced by Ser(19) thiophosphorylation. However, phosphorylation of Ser(19) or both Ser(19) and Thr(18) to comparable stoichiometries (0.5 mol of P(i)/mol of LC(20)) and similar levels of isometric force revealed differences in the rates of dephosphorylation and relaxation following removal of the stimulus: t(½) values for dephosphorylation were 83.3 and 560 s, and for relaxation were 560 and 1293 s, for monophosphorylated (Ser(19)) and diphosphorylated LC(20), respectively. We conclude that phosphorylation at Thr(18) decreases the rates of LC(20) dephosphorylation and smooth muscle relaxation compared with LC(20) phosphorylated exclusively at Ser(19). These effects of LC(20) diphosphorylation, combined with increased Ser(19) phosphorylation (Ca(2+)-independent), may underlie the hypercontractility that is observed in response to certain physiological contractile stimuli, and under pathological conditions such as cerebral and coronary arterial vasospasm, intimal hyperplasia, and hypertension.     

Intrasteric regulation of myosin light chain kinase: the pseudosubstrate prototope binds to the active site.

We previously proposed a molecular mechanism for the activation of smooth muscle myosin light chain kinase (smMLCK) by calmodulin (CaM). According to this model, smMLCK is autoinhibited in the absence of Ca2+/CaM due to the interaction of a pseudosubstrate prototope, contained within the CaM binding/regulatory region, with the active site of the enzyme. Binding of Ca2+/CaM releases the autoinhibition and allows access of the protein substrate to the active site of the enzyme, resulting in phosphorylation of the myosin light chains. We now provide direct experimental evidence that the pseudosubstrate prototope can associate with the active site. We constructed a smMLCK mutant in which the five-amino acid phosphorylation site of the myosin light chain substrate was inserted into the pseudosubstrate sequence of the CaM binding domain without disrupting the ability of the enzyme to bind Ca2+/CaM. We demonstrate that this mutant undergoes intramolecular autophosphorylation at the appropriate inserted serine residue in the absence of CaM and that this autophosphorylation activates the enzyme. Binding of Ca2+/CaM to the mutant enzyme stimulated myosin light chain substrate phosphorylation but strongly inhibited autophosphorylation, presumably by removing the pseudosubstrate from the active site. These results confirm that the pseudosubstrate sequence has access to the catalytic site and that the activation of the enzyme is accompanied by its removal from this position due to Ca2+/CaM binding as predicted by the model.     

Stretch activates myosin light chain kinase in arterial smooth muscle

Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol [32P]phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the [32P]phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in [32P]phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.     

Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells.

Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca2+. Pretreatment of cells with 1-[N-O-bis(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca2+]i but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca2+ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca2+ and potentiated myosin light chain phosphorylation. We propose that the Ca(2+)-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca2+ sensitivity of myosin light chain phosphorylation in smooth muscle.     

Regulation of Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II

The 63-kDa subunit, but not the 60-kDa subunit, of brain calmodulin-dependent cyclic nucleotide phosphodiesterase was phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II. When calmodulin was bound to the phosphodiesterase, 1.33 +/- 0.20 mol of phosphate was incorporated per mol of the 63-kDa subunit within 5 min with no significant effect on enzyme activity. Phosphorylation in the presence of low concentrations of calmodulin resulted in a phosphorylation stoichiometry of 2.11 +/- 0.21 and increased about 6-fold the concentration of calmodulin necessary for half-maximal activation of the phosphodiesterase. Peptide mapping analyses of complete tryptic digests of the 63-kDa subunit revealed two major (P1, P4) and two minor (P2, P3) 32P-peptides. Calmodulin-binding to the phosphodiesterase almost completely inhibited phosphorylation of P1 and P2 with reduced phosphorylation rates of P3 and P4, suggesting the affinity change of the enzyme for calmodulin may be caused by phosphorylation of P1 and/or P2. When Ca2+/calmodulin-dependent protein kinase II was added without prior autophosphorylation, there was no phosphorylation of the 63-kDa phosphodiesterase subunit or of the kinase itself in the presence of a low concentration of calmodulin, and with excess calmodulin the phosphodiesterase subunit was phosphorylated only at P3 and P4. Thus the 63-kDa subunit of phosphodiesterase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II and blocked by Ca2+/calmodulin binding to the subunit.     

Role of threonine-286 as autophosphorylation site for appearance of Ca2(+)-independent activity of calmodulin-dependent protein kinase II alpha subunit.

To confirm directly the role of Thr-286 as the autophosphorylation site responsible for the appearance of Ca2(+)-independent activity of Ca2+/calmodulin-dependent protein kinase II alpha subunit, we constructed two mutated cDNAs of Thr-286 to Pro or Ala using site-directed mutagenesis and introduced into Chinese hamster ovary cells. The mutant enzymes expressed in stable cell lines were partially purified and their catalytic properties were confirmed to be similar to those of wild-type kinase, except that the mutant kinase which were deprived of Thr-286 as an autophosphorylation site could not be converted to Ca2(+)-independent forms upon autophosphorylation. Other autophosphorylation sites of the mutants were essentially unchanged from those of the wild-type kinase and phosphorylation of such sites did not convert them to Ca2(+)-independent forms. The results indicate that Thr-286 is the only indispensable autophosphorylation site for the appearance of Ca2(+)-independent activity of calmodulin-dependent protein kinase II alpha subunit.     

Phosphorylation of smooth muscle myosin heavy and light chains. Effects of phorbol dibutyrate and agonists

A number of different protein kinases phosphorylate purified heavy chains or the 20-kDa light chain of smooth muscle myosin. The physiological significance of these phosphorylation reactions has been examined in intact smooth muscle. Myosin heavy chain was slightly phosphorylated (0.08 mol of phosphate/mol) under control conditions in bovine tracheal tissue. Treatment with carbachol, isoproterenol, or phorbol 12,13-dibutyrate resulted in no significant change. In contrast, heavy chain was phosphorylated to 0.30 mol of phosphate/mol of heavy chain in tracheal smooth muscle cells in culture. This value increased significantly with ionomycin treatment. In control tissues, 9% of the light chain was monophosphorylated with 32P in the serine site phosphorylated by myosin light chain kinase. Carbachol (0.1 microM) alone resulted in contraction and 42% monophosphorylated light chain with 32P only in the serine site phosphorylated by myosin light chain kinase. Similarly, stimulation with histamine, 5-hydroxytryptamine, or KCl resulted in 32P incorporation into only the myosin light chain kinase serine site. Phorbol 12,13-dibutyrate (1 microM) alone resulted in 22% monophosphorylated light chain. However, only 25% of the 32P was in the myosin light chain kinase serine site, whereas 75% was in a serine site phosphorylated by protein kinase C. Phorbol 12,13-dibutyrate plus carbachol resulted in 27% monophosphorylated light chain; 75% of the 32P was in the myosin light chain kinase serine site, with the remainder in the protein kinase C serine site. These results indicate that phorbol esters act to increase phosphorylation of myosin light chain by protein kinase C. However, receptor-mediated stimulation or depolarization leading to tracheal smooth muscle contraction results in phosphorylation of myosin light chain by myosin light chain kinase alone.     

The involvement of Ca2+ and myosin light chain kinase in collagen-induced platelet activation.

In this study we have used several complementary biochemical and immunological techniques to examine the involvement of Ca2+ and myosin light chain kinase in collagen-induced platelet activation. Our results indicate that collagen stimulates a rapid influx of external Ca2+ (within the first 1-5 min of treatment) which is followed by phosphorylation of myosin light chains (within 10 min of treatment) and granule secretion (within 15 min of treatment). In addition, we have found that certain Ca2+ channel entry blockers (e.g. nifedipine and bepridil) or calmodulin antagonists (e.g. W-7) specifically inhibit collagen-induced Ca2+ influx, myosin light chain phosphorylation and subsequent granule secretion. These data suggest that Ca2+/calmodulin-dependent myosin light chain kinase-mediated myosin light chain phosphorylation is necessary for regulating the actomyosin-related contractility required for normal platelet function.