Abstract
It has been observed that the activity of Ca2+-calmodulin (CaM)-dependent protein kinase I is enhanced up to 50-fold by its phosphorylation in vitro by a distinct CaM kinase I kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164). It has, however, been unclear whether this event represents an acute form of cellular regulation. We demonstrate here the phosphorylation and activation of CaM kinase I in PC12 pheochromocytoma cells in response to elevation of intracellular Ca2+. Treatment of PC12 cells with the Ca2+-ionophore, ionomycin, or with a depolarizing concentration of KCl, led to rapid, biphasic phosphorylation of CaM kinase I and to increases in CaM kinase I activity of 5.1- and 7. 3-fold, respectively. Depolarization-induced activation of CaM kinase I was reduced by approximately 80% by blockade of Ca2+ influx through L-type voltage-dependent Ca2+ channels and completely abolished by removal of extracellular Ca2+. The ability of PC12 cell CaM kinase I to be phosphorylated and activated by purified CaM kinase I kinase in vitro was markedly reduced by prior depolarization of the cells, consistent with intracellular phosphorylation and activation of CaM kinase I by CaM kinase I kinase. These results demonstrate the existence in PC12 cells of a CaM kinase I cascade, the function of which may be to sensitize cells to signal-induced elevations of intracellular Ca2+.
Highlights
Control of cellular function by extracellular signal-induced elevation of intracellular Ca2ϩ is a common theme in biological systems [1]
We report here, using the PC12 pheochromocytoma cell line, that a CaMKI cascade exists in living cells and that initiation of this cascade can occur in response to either a Ca2ϩ-ionophore, ionomycin, or to a membrane depolarizationinduced Ca2ϩ influx through L-type voltage-dependent Ca2ϩ channels
Unlike mitogen-activated protein kinase (MAPK) and MAPKKs, CaMKI exhibits, in addition to regulation by phosphorylation, absolute dependence of its activity upon direct, allosteric activation [7, 16], a dependence completely retained by the phosphorylated enzyme [12, 17]
Summary
CaM, calmodulin; CaM kinases, Ca2ϩCaM-dependent protein kinases; CaMKIK, Ca2ϩ-CaM-dependent protein kinase I kinase; synapsin site-1 peptide, NYLRRRLSDSNF, synthetic peptide based on phosphorylation site 1 [33] of synapsin I; CC76, anti-CaMKI antibody; MAPK, mitogen-activated protein kinase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline. Activation results from the phosphorylation by CaMKIK of a single residue, Thr-177 [7, 20, 21] located in the “activation loop” of CaMKI [22] This residue is at a position equivalent to the activating phosphorylation sites in a number of other protein kinases [23], including those for which upstream kinase kinases have been identified, such as members of the mitogen-activated protein kinase (MAPK) and cyclindependent protein kinase families (24 –29). While these data predict that the phosphorylation and activation of CaMKI by CaMKIK may occur in vivo, there has, to date, been no experimental evidence for the existence of such a cascade in living cells. We report here, using the PC12 pheochromocytoma cell line, that a CaMKI cascade exists in living cells and that initiation of this cascade can occur in response to either a Ca2ϩ-ionophore, ionomycin, or to a membrane depolarizationinduced Ca2ϩ influx through L-type voltage-dependent Ca2ϩ channels
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