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.     

Growth hormone stimulation of amino acid transport and utilization by the perfused rat liver.

The effects of growth hormone, administered in vivo or added in vitro, on amino acid transport and utilization have been studied in perfused livers of normal and hypophysectomized rats. A perfusion system employing a nonrecirculating medium was used in all of the studies. Two nonmetalbolizable amino acid analogues, alpha-aminoisobutyric acid (AIB) and 1-aminocyclopentane carboxylic acid (cycloleucine) were used to study transport. Accumulation of AIB increased linearly over a 60-min perfusion period, reaching distribution ratios of between 1 and 2 for both groups of animals. Treatment of both normal and hypophysectomized rats with growth hormone 60 min prior to the start of perfusion increased AIB distribution ratios by up to 84 and 108%, respectively. Accumulation of cycloleucine was linear for only about 20 min of perfusion and then plateaued. Steady state distribution ratios of this analogue ranged between 1 and 2 for both groups of animals. Growth hormone treatment had no apparent effect on the time necessary to reach these steady state levels, but significantly increased them in livers of both normal and hypophysectomized rats by 16 and 42%, respectively. Studies designed to analyze the kinetic properties of these hormone effects revealed that growth hormone treatment caused 2-fold i-crease in the maximum velocities of both the AIB and cycloleucine transport systems. The substrate concentration for half-maximal transport velocity was increased slightly for both systems by growth hormone. Direct effects of growth hormone were demonstrated in studies where livers of hypophysectomized rats were perfused under conditions simulationg those of experiments in which the hormone was administered in vivo. Following an initial 45-min period of perfusion the medium during the 20 min. Growth hormone added to the medium during the entire 65-min perfusion at a concentration of 1 mug per ml caused a 30% increase in the cycloleucine distribution ratio. Under similar experimental conditions growth hormone directly stimulated three hepatic pathways of amino acid utilization: (a) incorporation of [14C]valine into protein, (b) urea formation and (c) conversion of 14-C-amino-acids to labeled glucose. Intracellular concentrations of seven amino acids, including threonine, serine, proline, glycine, alanine, lysine, and arginine, were increased significantly in livers perfused with medium containing growth hormone...     

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 M) 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 M); lower concentrations of ionomycin as well as stimulation with angiotensin II (1 M) or ATP (100 M) 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.     

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.     

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.     

Tissue-specific regulation of protein synthesis by insulin and free fatty acids

The purpose of the study described herein was to investigate how the mammalian target of rapamycin (mTOR)-signaling pathway and eukaryotic initiation factor 2B (eIF2B) activity, both having key roles in the translational control of protein synthesis in skeletal muscle, are regulated in cardiac muscle of rats in response to two different models of altered free fatty acid (FFA) and insulin availability. Protein synthetic rates were reduced in both gastrocnemius and heart of 3-day diabetic rats. The reduction was associated with diminished mTOR-mediated signaling and eIF2B activity in the gastrocnemius but only with diminished mTOR signaling in the heart. In response to the combination of acute hypoinsulinemia and hypolipidemia induced by administration of niacin, protein synthetic rates were also diminished in both gastrocnemius and heart. The niacin-induced changes were associated with diminished mTOR signaling and eIF2B activity in the heart but only with decreased mTOR signaling in the gastrocnemius. In the heart, mTOR signaling and eIF2B activity correlated with cellular energy status and/or redox potential. Thus FFAs may contribute to the translational control of protein synthesis in the heart but not in the gastrocnemius. In contrast, insulin, but not FFAs, is required for the maintenance of protein synthesis in the gastrocnemius.     

Substrate specificity of liver calmodulin-dependent glycogen synthase kinase

A number of proteins were tested as potential substrates for purified rabbit liver calmodulin-dependent glycogen synthase kinase. It was found that liver phenylalanine hydroxylase and several brain proteins including tyrosine hydroxylase, microtubule-associated protein 2, and synapsin I were readily phosphorylated. Brain tubulin was very poorly phosphorylated. These results suggest that calmodulin-dependent glycogen synthase kinase may be a more general protein kinase involved in the regulation of several cellular Ca²⁺-dependent functions.     

Purification and characterization of rabbit liver calmodulin-dependent glycogen synthase kinase

A rabbit liver cAMP-independent glycogen synthase kinase has been purified 4500-fold to a specific activity of 2.23 mumol of 32P incorporated per min per mg of protein using ion exchange chromatography on DEAE-Sephacel and phosphocellulose, gel filtration chromatography on Sepharose 6B, and affinity chromatography on calmodulin-Sepharose. This synthase kinase, which was completely dependent on the presence of calmodulin (apparent K0.5 = 0.1 microM) and calcium for activity, also catalyzed the phosphorylation of purified smooth muscle myosin light chain but not of smooth muscle myosin. Using 0.5 mM ATP, a maximal rate of phosphorylation of glycogen synthase was achieved in the presence of 10 mM magnesium acetate with a pH optimum of 7.8. Gel filtration experiments indicated a Stokes radius of about 70 A and sucrose density gradient centrifugation data gave a sedimentation coefficient of 10.6 S. A molecular weight of approximately 300,000 was calculated. A definitive subunit structure was not determined, but major bands observed after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate corresponded to a doublet at 50,000 to 53,000. The calmodulin-dependent glycogen synthase kinase incorporated about 1 mol of 32P per mol of synthase subunit into sites 2 and 1b associated with a decrease in the synthase activity ratio from 0.8 to about 0.4. The calmodulin-dependent glycogen synthase kinase may mediate the effects of alpha-adrenergic agonists, vasopressin, and/or angiotensin II on glycogen synthase in liver.