Ca2+-induced redistribution of Ca2+/calmodulin-dependent protein kinase II associated with an endoplasmic reticulum stress response in vascular smooth muscle.

The relation between CaM kinase II activity and high Ca2+-mediated stress responses was studied in cultured vascular smooth muscle cells. Treatment with ionomycin (1 microM) for 5 min caused a significant loss of CaM kinase II activity in whole cell homegenates and prominent vesiculation of the endoplasmic reticulum (ER). Similar losses of CaM kinase II activity were observed in the soluble lysate as assessed by activity measurements and Western blotting. Examination of the post-lysate particulate fraction showed that the loss of CaM kinase II from the soluble lysate was accompanied by a redistribution of CaM kinase II to this fraction. The ionomycin-mediated response was limited to this concentration (1 microM); lower concentrations of ionomycin as well as stimulation with angiotensin II (1 microM) orATP (100 microM) did not cause a shift in CaM kinase II distribution. Treatment with neither the CaM kinase II inhibitor KN-93 nor the phosphatase inhibitor okadaic acid altered the ionomycin-induced redistribution indicating that CaM kinase II activation and/or phosphorylation was not part of the mechanism. The response, however, was eliminated when the cells were treated in Ca2+-free medium. Washout of ionomycin led to only a partial restoration of the kinase activity in the soluble fraction after 10 min. Immunofluorescence microscopy of resting cells indicated colocalization of antibodies to CaM kinase II and an ER protein marker. ER vesiculation induced by ionomycin coincided with a parallel redistribution of CaM kinase II and ER marker proteins. These data link ionomycin-induced ER restructuring to a progressive redistribution of CaM kinase II protein to an insoluble particulate fraction and loss of cellular CaM kinase II activity. We propose that redistribution of CaM kinase II and loss of cellular activity are components of a common Ca2+-overload induced cellular stress response in cells.

A role for Ca2+/calmodulin-dependent protein kinase II in the mitogen-activated protein kinase signaling cascade of cultured rat aortic vascular smooth muscle cells.

Exposure of cultured rat aortic vascular smooth muscle (VSM) cells to the Ca2+ ionophore ionomycin produced an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) activity that was maximal between 2 and 5 minutes but then declined to basal values within 20 minutes of stimulation. Elevation of [Ca2+]i in VSM cells leads to an even more rapid activation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II); thus, it was postulated that the Ca(2+)-dependent component of ERK1/2 activation was mediated by CaM kinase II. Transient ERK1/2 activation by ionomycin was almost completely abolished by pretreating cells with 30 mumol/L KN-93, a CaM kinase II inhibitor. Treatment of cells with KN-93 did not antagonize the ability of ionomycin to mobilize intracellular Ca2+ but prevented CaM kinase II and ERK1/2 activation with almost identical potencies. Consistent with a role for Ca2+ and calmodulin in intracellular Ca(2+)-induced activation of ERK, cells pretreated with calmodulin inhibitors (W-7 or calmidazolium) exhibited an attenuated ERK response to ionomycin. ERK1/2 activation in response to phorbol esters and platelet-derived growth factor were not significantly affected by KN-93, whereas the response to angiotensin II and thrombin were attenuated by 60% and 40%, respectively. Transient expression of wild-type delta 2 CaM kinase II in COS-7 cells resulted in increased ERK2 activity, whereas coexpression of wild-type and a kinase-negative mutant resulted in a diminution of this response. These data suggest that regulation of cellular responses by Ca(2+)-dependent pathways in VSM cells may be mediated in part by CaM kinase II-dependent activation of ERK1/2.

Novel Ca2+/calmodulin-dependent protein kinase II gamma-subunit variants expressed in vascular smooth muscle, brain, and cardiomyocytes.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) gamma-subunits were cloned from a porcine aortic smooth muscle cDNA library resulting in identification of alternatively spliced CaM kinase II gammaB- and gammaC-subunits and a novel gamma-subunit variant predicted to encode a 60.2-kDa polypeptide, which was designated the gammaG-subunit. A clone predicted to encode a 62. 2-kDa gamma-subunit, designated as gammaE, was isolated with a variable domain structure similar to a gammaB-subunit but with a 114-nucleotide insertion in the conserved "association" domain of CaM kinase II subunits. A full-length gammaE-subunit construct expressed in COS cells resulted in multimeric CaM kinase II holoenzymes (470 kDa) with activation and autoregulatory properties similar to expressed holoenzymes composed of gammaB-, gammaC-, or gammaG-subunits. Expression of gammaE and related gamma-subunit mRNAs containing the 114-base insertion was documented in porcine tissues by reverse transcriptase-polymerase chain reaction. CaM kinase II subunits containing the 38-amino acid insert were identified by Western analysis of partially purified CaM kinase II from carotid arterial smooth muscle and brain using a sequence-specific anti-peptide antibody. Immunoprecipitations of tissue homogenates indicated a comparatively high level of expression of subunits containing the insert in brain and provided evidence for their co-assembly with other more abundant subunits into CaM kinase II heteromultimers. Our analyses indicate the following patterns of gamma-subunit expression: vascular smooth muscle, gammaB > gammaC > gammaE,G; heart, gammaB > gammaE,C > gammaG; brain, gammaE and related subunits >> gammaA,B,C,G.

In situ Ca2+ dependence for activation of Ca2+/calmodulin-dependent protein kinase II in vascular smooth muscle cells.

Activation of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM kinase II) and development of the Ca2+/CaM-independent (autonomous) form of the kinase was investigated in cultured vascular smooth muscle (VSM) cells. Within 15 s of ionomycin (1 microM) exposure 52.7 +/- 4.4% of the kinase became autonomous, a response that was partially maintained for at least 10 min. This correlated with 32P phosphorylation of CaM kinase II delta-subunits in situ and was abolished by pretreatment with the CaM kinase II inhibitor KN-93. The in situ Ca2+ dependence for generating autonomous CaM kinase II was determined in cells selectively permeabilized to Ca2+ and depleted of sarcoplasmic reticulum Ca2+ by pretreatment with thapsigargin. Analysis of the resulting curve revealed an EC50 (concentration producing 50% of maximal response) of 692 +/- 28 nM [Ca2+]i, a maximum of 68 +/- 2% of the total activity becoming autonomous reflecting nearly complete activation of CaM kinase II and a Hill slope of 3, indicating a highly cooperative process. Based on this dependence and measured [Ca2+]i responses in intact cells, increases in autonomous activity stimulated by angiotensin II, vasopressin and platelet-derived growth factor-BB (4.6-, 2-, and 1.7-fold, respectively) were unexpectedly high. In intact cells stimulated by ionomycin, the correlation between autonomous activity and [Ca2+]i resulted in a parallel curve with an EC50 of 304 +/- 23 nM [Ca2+]i. This apparent increase in Ca2+ sensitivity for generating autonomous activity in intact VSM cells was eliminated by thapsigargin pretreatment. We conclude that alteration of [Ca2+]i over a physiological range activates CaM kinase II in VSM and that this process is facilitated by release of Ca2+ from intracellular pools which initiates cooperative autophosphorylation and consequent generation of autonomous CaM kinase II activity.

Identification of novel isoforms of the delta subunit of Ca2+/calmodulin-dependent protein kinase II. Differential expression in rat brain and aorta.

Two novel isoforms of the Ca2+/calmodulin-dependent protein kinase II delta subunit were detected in rat aorta. Identification of the subunits was based on two independent lines of evidence, i.e. detection by immunoblotting of differently sized delta subunits and DNA sequence analysis of partial cDNA clones of the kinase. Cytosolic extracts from rat brain, aorta, and cultured aortic cells were analyzed by Western blotting using a delta subunit-specific antipeptide antibody. Aortic extracts demonstrated a single 53-kDa cross-reactive band approximately 7 kDa smaller than the cross-reactive band seen in brain. To ascertain the structural basis for this difference, reverse-transcribed RNAs from rat aorta and brain were analyzed by polymerase chain reaction (PCR), and the PCR fragments were cloned and sequenced. When aortic cDNA was analyzed with a primer pair that spanned the known variable region of the brain kinase subunit, the amplified PCR products were smaller than the major product obtained from brain cDNA. The aortic PCR product was cloned and sequenced and found to represent two novel subunit sequences, designated delta 2 and delta 3 to distinguish them from the previously described delta sequence (now called delta 1) from brain. delta 2 was identical to the predicted delta 1 sequence except for a deletion of 102 base pairs (bp). This deletion corresponded to nearly the entire variable domain. In the sequence of delta 3, this 102-bp region was replaced by a sequence of 33 bp that had 79% nucleotide sequence identity to a portion of the gamma subunit variable domain. A fourth form of the delta subunit (delta 4) was identified in rat skeletal muscle. The delta 4 isoform was characterized by the deletion of a 42-bp sequence identical to the 42 bp at the 3' end of the 102-bp deletion of delta 2. Reverse-transcription PCR analysis of additional rat tissues indicated that alternatively spliced variants of the delta subunit of Ca2+/calmodulin-dependent protein kinase II are expressed in a tissue-specific pattern.

Activation of protein kinase C isozymes by contractile stimuli in arterial smooth muscle.

Protein kinase C (PKC) has been proposed to be involved in the regulation of vascular smooth muscle (VSM) contractile activity. However, little is known in detail about the activation of this kinase or specific isozymes of this kinase by contractile stimuli in VSM. As an index of PKC activation, Ca(2+)- and phospholipid-dependent histone IIIS kinase activity was measured in the particulate fraction from individual strips of isometrically contracting carotid arterial smooth muscle. Phorbol 12,13-dibutyrate (PDB) increased PKC activity in the particulate fraction (155% over resting value by 15 min) with a time course which paralleled or preceded force development. Stimulation with the agonist histamine (10(-5) M) resulted in rapid increases in both force and particulate fraction PKC activity which was maximal by 2 min (increase of 139%) and partially sustained over 45 min (increase of 41%). KCl (109 mM), which evokes a sustained contractile response, caused a slow increase (124% by 45 min) in particulate fraction PKC activity. No significant increases in activator-independent histone kinase activity were observed in response to any stimulus tested. PKC alpha and PKC beta were identified as the principal Ca2+/phospholipid-dependent PKC isozymes expressed in this tissue. In unstimulated arterial tissue, the ratio of immunodetectable isozyme content (alpha:beta) was estimated to be 1:1 in the particulate and 1.5:1 in the cytosolic fractions. Upon stimulation with each of the three contractile stimuli, particulate fraction PKC content assessed by immunoblotting increased with a time course and to an extent comparable to the observed changes in PKC activity. There was no evidence of differential regulation of the PKC alpha or -beta isozymes by PDB compared to the other contractile stimuli. These results indicate that diverse contractile stimuli are capable of tonically activating PKC in preparations of functional smooth muscle, and are consistent with a functional role for PKC alpha and/or -beta in the regulation of normal smooth muscle contractile activity.

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.

Proteolytic activation of calcium/calmodulin-dependent protein kinase II: Putative function in synaptic plasticity.

Rat forebrain Ca(2+)/calmodulin-dependent protein kinase II (CaM-kinase II) in isolated postsynaptic densities (PSD) was subjected to limited proteolysis with chymotrypsin or mu-calpain, a Ca(2+)-dependent protease. Incubation of the kinase with either protease resulted in a three- to fivefold enhancement of total kinase activity and solubilization of Ca(2+)/calmodulin (CaM)-independent activity from the PSD. Maximal enhancement of CaM-kinase II activity was observed when autophosphorylated or Ca(2+)/CaM-bound forms of the enzyme were proteolyzed. Analysis of the proteolytic products by Western blotting with a polyclonal antibody raised against soluble CaM-kinase II indicated that both proteases generated several immunoreactive fragments between 21 and 32 kDa. However, unlike chymotrypsin, mu-calpain degraded only a small fraction of the intact kinase subunits. (125)I-labeled CaM overlays indicated a major CaM-binding fragment of approximately 23 kDa in mu-calpain digests of purified cytosolic CaM-kinase II. This fragment was also shown to contain the regulatory autophosphorylation site (Thr-286(alpha)/287(beta)) of the kinase. Immunoblotting with antibody to the catalytic domain of the kinase indicated that there was a single active fragment of approximately 30 kDa in the mu-calpain digests. Analysis of the crude digests on a Superose-6 FPLC column also indicated that the Ca(2+)/CaM-independent activity resided in a fragment of approximately 30 kDa. This catalytic fragment did not bind to CaM-Sepharose. Thus, mu-calpain appears to cleave CaM-kinase 11 into a 30-kDa catalytic domain fragment and a 23-kDa regulatory domain fragment. A putative mechanism for persistent regulation of synaptic events by such a proteolytic activation of CaM-kinase 11 is discussed.

Regulatory properties of calcium/calmodulin-dependent protein kinase II in rat brain postsynaptic densities.

Calcium/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) contained within the postsynaptic density (PSD) was shown to become partially Ca2+-independent following initial activation by Ca2+/CaM. Generation of this Ca2+-independent species was dependent upon autophosphorylation of both subunits of the enzyme in the presence of Mg2+/ATP/Ca2+/CaM and attained a maximal value of 74 +/- 5% of the total activity within 1-2 min. Subsequent to the generation of this partially Ca2+-independent form of PSD CaM-kinase II, addition of EGTA to the autophosphorylation reaction resulted in further stimulation of 32PO4 incorporation into both kinase subunits and a loss of stimulation of the kinase by Ca2+/CaM. Examination of the sites of Ca2+-dependent autophosphorylation by phosphoamino acid analysis and peptide mapping of both kinase subunits suggested that phosphorylation of Thr286/287 of the alpha- and beta-subunits, respectively, may be responsible for the transition of PSD CaM-kinase II to the Ca2+-independent species. A synthetic peptide 281-309 corresponding to a portion of the regulatory domain (residues 281-314) of the soluble kinase inhibited syntide-2 phosphorylation by the Ca2+-independent form of PSD CaM-kinase II (IC50 = 3.6 +/- 0.8 microM). Binding of Ca2+/CaM to peptide 281-309 abolished its inhibitory property. Phosphorylation of Thr286 in peptide 281-309 also decreased its inhibitory potency. These data suggest that CaM-kinase II in the PSD possesses regulatory properties and mechanisms of activation similar to the cytosolic form of CaM-kinase II.

Regulatory domain of calcium/calmodulin-dependent protein kinase II. Mechanism of inhibition and regulation by phosphorylation.

Regulatory mechanisms of rat brain Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) were probed using a synthetic peptide (CaMK-(281-309] corresponding to residues 281-309 (alpha-subunit) which contained the calmodulin (CaM)-binding and inhibitory domains and also the initial autophosphorylation site (Thr286). Kinetic analyses indicated that inhibition of a completely Ca2+/CaM-independent form of CaM-kinase II by CaMK-(281-309) was noncompetitive with respect to peptide substrate (syntide-2) but was competitive with respect to ATP. Interaction of CaMK-(281-309) with the ATP-binding site was independently confirmed since inactivation of proteolyzed CaM-kinase II by phenylglyoxal (t1/2 = 7 min) was blocked by ATP analog plus Mg2+ or by CaMK-(281-309). In the presence of Ca2+/CaM, CaMK-(281-309) no longer protected against phenylglyoxal inactivation, consistent with our previous observations (Colbran, R.J., Fong, Y.-L., Schworer, C.M., and Soderling, T.R. (1988) J. Biol. Chem. 263, 18145-18151) that binding of Ca2+/CaM to CaMK-(281-309) 1) blocks its inhibitory property, and 2) enhances its phosphorylation at Thr 286. The present study also showed that phosphorylation of CaMK-(281-309) decreased its inhibitory potency at least 10-fold without affecting its Ca2+/CaM-binding ability. Thus, CaM-kinase II is inactive in the absence of Ca2+/CaM because an inhibitory domain within residues 281-309 interacts with the catalytic domain and blocks ATP binding. Autophosphorylation of Thr286 results in a Ca2+/CaM-independent form of the kinase by disrupting the inhibitory interaction with the catalytic domain.

Regulatory interactions of the calmodulin-binding, inhibitory, and autophosphorylation domains of Ca2+/calmodulin-dependent protein kinase II.

Two peptide analogs of Ca2+/calmodulin-dependent protein kinase II (CaMK-(peptides)) were synthesized and used to probe interactions of the various regulatory domains of the kinase. CaMK-(281-289) contained only Thr286, the major Ca2+-dependent autophosphorylation site of the kinase (Schworer, C. M., Colbran, R. J., Keefer, J. R. & Soderling, T. R. (1988) J. Biol. Chem. 263, 13486-13489), whereas CaMK-(281-309) contained Thr286 together with the previously identified calmodulin binding and inhibitory domains (Payne, M. E., Fong, Y.-L., Ono, T., Colbran, R. J., Kemp, B. E., Soderling, T. R. & Means, A. R. (1988) J. Biol. Chem. 263, 7190-7195). CaMK-(281-309), but not CaMK-(281-289), bound calmodulin and was a potent inhibitor (IC50 = 0.88 +/- 0.7 microM using 20 microM syntide-2) of exogenous substrate (syntide-2 or glycogen synthase) phosphorylation by a completely Ca2+/calmodulin-independent form of the kinase generated by limited proteolysis with chymotrypsin. This inhibition was completely relieved by the inclusion of Ca2+/calmodulin in excess of CaMK-(281-309) in the assays. CaMK-(281-289) was a good substrate (Km = 11 microM; Vmax = 3.15 mumol/min/mg) for the proteolyzed kinase whereas phosphorylation of CaMK-(281-309) showed nonlinear Michaelis-Menton kinetics, with maximal phosphorylation (0.1 mumol/min/mg) at 20 microM and decreased phosphorylation at higher concentrations. The addition of Ca2+/calmodulin to assays stimulated the phosphorylation of CaMK-(281-309) by the proteolyzed kinase approximately 10-fold but did not affect the phosphorylation of CaMK-(281-289). A model for the regulation of Ca2+/calmodulin-dependent protein kinase II is proposed based on the above observations and results from other laboratories.

Ca2+/calmodulin-dependent protein kinase II. Identification of a regulatory autophosphorylation site adjacent to the inhibitory and calmodulin-binding domains.

Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) autophosphorylated under limiting conditions (7 microM [gamma-32P]ATP, 500 microM magnesium acetate, 4 degrees C) was analyzed by CNBr cleavage and peptide mapping to determine the site of autophosphorylation that brings about transition of the kinase to the Ca2+-independent form. Reverse phase high performance liquid chromatography (HPLC) (C3) revealed one major CN-Br 32P-peptide (CB1) that eluted at about 6% propanol. This peptide contained [32P]threonine, but almost no [32P]serine, and migrated as a single band (Mr = 3000-3500) in polyacrylamide gels run in the presence of urea and sodium dodecyl sulfate. The properties of CB1 were compared to the properties of a 26-residue synthetic peptide containing the CaM-binding and inhibitory domains as well as a consensus phosphorylation sequence (-Arg-Gln-Glu-Thr-) of rat brain CaM-kinase II (residues 282-307 and 283-308 of the alpha and beta subunits, respectively). CB1 and the synthetic peptide comigrated in urea/sodium dodecyl sulfate gels, co-eluted from reverse phase HPLC (C3 and C18) and from Sephadex G-50, and exhibited Ca2+-dependent calmodulin-binding properties. When the two peptides were subjected to automated Edman sequence analysis, both exhibited a burst of 32P release at cycle 5, which is consistent with the expected amino-terminal sequence of the two peptides, i.e. His-Arg-Gln-Glu-Thr(PO4)-. These findings indicate that autophosphorylation of Thr286 (alpha subunit) and Thr287 (beta subunit) is responsible for transition of CaM-kinase II to the Ca2+-independent form.

Substrate specificity of Ca2+/CaM-dependent multifunctional protein kinases: comparison of isoenzymes from brain, liver and skeletal muscle.

Ca2+/CaM-dependent multifunctional protein kinase isoenzymes from brain, skeletal muscle and liver were compared by their phosphorylation of a number of protein substrates. Under the conditions of assay, the three isoenzymes demonstrated rapid phosphorylation of synapsin I and glycogen synthase. In contrast, rates of phosphorylation of pyruvate kinase and phenylalanine hydroxylase were almost two orders of magnitude slower. Differences in phosphorylation specifically of the latter two substrates was also observed among the three protein kinases. Phosphorylation by Ca2+/CaM-dependent protein kinases was contrasted with cAMP-dependent protein kinase, which phosphorylates these proteins in vitro and in vivo. The potential role of Ca2+/CaM-dependent multifunctional protein kinases in the Ca2+-dependent phosphorylation of these substrates is discussed.

Identification of membrane-bound calcium, calmodulin-dependent protein kinase II in canine heart.

Phospholamban, the putative regulatory proteolipid of the Ca2+/Mg2+ ATPase in cardiac sarcoplasmic reticulum, was selectively phosphorylated by a Ca2+/calmodulin (CaM)-dependent protein kinase associated with a cardiac membrane preparation. This kinase also catalyzed the phosphorylation of two exogenous proteins known to be phosphorylated by the multifunctional Ca2+/CaM-dependent protein kinase II (Ca2+/CaM-kinase II), i.e., smooth muscle myosin light chains and glycogen synthase a. The latter protein was phosphorylated at sites previously shown to be phosphorylated by the purified multifunctional Ca2+/CaM-kinase II from liver and brain. The membrane-bound kinase did not phosphorylate phosphorylase b or cardiac myosin light chains, although these proteins were phosphorylated by appropriate, specific calmodulin-dependent protein kinases added exogenously. In addition to phospholamban, several other membrane-associated proteins were phosphorylated in a calmodulin-dependent manner. The principal one exhibited a Mr of approximately 56,000, a value similar to that of the major protein (57,000) in a partially purified preparation of Ca2+/CaM-kinase II from the soluble fraction of canine heart that was autophosphorylated in a calmodulin-dependent manner. These data indicate that the membrane-bound, calmodulin-dependent protein kinase that phosphorylates phospholamban in cardiac membranes is not a specific calmodulin-dependent kinase, but resembles the multifunctional Ca2+/CaM-kinase II. Our data indicate that this kinase may be present in both the particulate and soluble fractions of canine heart.

Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II. Effects on total and Ca2+-independent activities and kinetic parameters.

Incubation of purified rat brain Ca2+/calmodulin-dependent protein kinase II for 2 min in the presence of Ca2+, calmodulin (CaM), Mg2+, and ATP converted the kinase from a completely Ca2+-dependent kinase to a substantially Ca2+-independent form with little loss of total activity. Subsequent addition of EGTA to the autophosphorylation reaction enhanced further autophosphorylation of the kinase which was associated with a suppression of total kinase activity to the Ca2+-independent value. Protein phosphatase 1 rapidly increased the suppressed total activity back to the control value and slowly decreased the Ca2+-independent activity. Kinetic analysis showed that the kinase not previously autophosphorylated had a Km for the synthetic peptide syntide-2 of 7 microM and Vmax of 9.8 mumol/min/mg when assayed in the presence of Ca2+ and CaM. The partially Ca2+-independent species, assayed in the presence of EGTA, had a Km of 21 microM and Vmax of 6.0. In the presence of Ca2+ and CaM the Km decreased and the Vmax increased to approximately control nonphosphorylated values. The completely Ca2+-independent form generated by sequential autophosphorylation first in the presence of Ca2+ and then EGTA had similar kinetic parameters to the partially independent species when assayed in the presence of EGTA, but addition of Ca2+ and CaM (up to 1 mg/ml) had little effect. These results suggest that separate autophosphorylation sites in the Ca2+/CaM-dependent protein kinase II are associated with formation of Ca2+-independent activity and suppression of total activity.

Phosphorylation of liver pyruvate kinase by Ca++/calmodulin-dependent protein kinase: characterization of two phosphorylation sites.

Rat liver pyruvate kinase is phosphorylated by calcium/calmodulin-dependent protein kinase II at serine and threonine residues in a 3-4 kDa CNBr fragment located near the amino terminus. The two sites of phosphorylation were separated by reverse-phase HPLC of a thermolysin digest. Sequence analysis established the sites of phosphorylation as follows: Leu-Arg-Arg-Ala-Ser(PO4)-Val-Ala-Gln-Leu-Thr(PO4)-Gln-Glu.

Reversible generation of a Ca2+-independent form of Ca2+(calmodulin)-dependent protein kinase II by an autophosphorylation mechanism.

The Ca2+(calmodulin (CaM))-dependent protein kinase II, purified from either rabbit liver or rat brain, was preincubated under conditions that are known to promote its autophosphorylation. When kinase activity was assayed after this preincubation, it was observed that excess EGTA could block no more than 40-60% of the total Ca2+- and CaM-dependent activity compared to 95% inhibition by EGTA prior to preincubation. In the EGTA assay, free Ca2+ was calculated to be less than 1 nM; therefore, this activity was designated Ca2+-independent activity. Formation of this Ca2+-independent form of the kinase was shown to be associated with autophosphorylation based on the following observations: (a) it required the presence of Ca2+, CaM, and ATP; (b) the ATP analogs adenylyl imidodiphosphate and adenylyl methylenediphosphate could not substitute for ATP; (c) generation of the independent form was associated with incorporation of phosphate into the kinase; and (d) addition of protein phosphatase partially dephosphorylated the kinase and restored its Ca2+ dependence. This phenomenon may be of physiological importance because it would prolong the effects of extracellular signals that only transiently increase the intracellular Ca2+ level.