Rapid activation of calmodulin-dependent protein kinase III in mitogen-stimulated human fibroblasts. Correlation with intracellular Ca2+ transients.
Growth-arrested human fibroblasts respond to mitogenic stimulation with a rapid, transient increase in cytoplasmic free Ca2+. This event may be crucial to the activation of Na/H exchange and subsequent DNA synthesis. Previous studies have implicated calmodulin (CaM) as a possible mediator of the effects of Ca2+ on these processes. here, we demonstrate that a specific CaM-dependent protein kinase (CaM-PK) system is rapidly activated in quiescent fibroblasts stimulated by a variety of mitogens. Cytoplasmic extracts of two human fibroblast cell types contained a major Ca2+-stimulated phosphoprotein of Mr 100,000 and pI 6.8 (Mr 100,000). This protein was shown by peptide mapping and immunological criteria to be identical to the prominent CaM-PK III substrate previously identified in a number of mammalian cells and tissues (Palfrey, H. C. (1983) FEBS Lett. 157, 183-190; Nairn, A.C., Bhagat, B., and Palfrey, H.C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7939-7943). Stimulation of 32P-labeled serum-deprived fibroblasts with serum, individual growth factors (bradykinin, vasopressin, and epidermal growth factor), or Ca2+ ionophores resulted in a rapid 2- to 10-fold increase in the phosphorylation of Mr 100,000 as determined by immunoprecipitation using polyclonal antibodies. With serum or individual growth factors, the effect peaked at 0.5-1 min then declined back to base line within 5 min. Time course studies showed that the phosphorylation state of Mr 100,000 closely paralleled but lagged slightly behind the Ca2+ transient (measured with fura-2). Thus, dephosphorylation of Mr 100,000 must follow shortly after Ca2+ levels begin to decline. The effects of serum, bradykinin, and vasopressin on both the rise in intracellular Ca2+ and the phosphorylation of Mr 100,000 were independent of external Ca2+, whereas the effects of epidermal growth factor and A23187 required external Ca2+. Phosphorylation of Mr 100,000 in intact cells took place on threonine residues, a major portion occurring in the same major phosphopeptide found in the protein labeled in vitro. These results show that mitogenic activation of human fibroblasts leads to the binding of Ca2+ to CaM and the subsequent activation of CaM-dependent processes.
D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002460:Cell Line; D054736:Elongation Factor 2 Kinase; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D014667:Vasopressins / Q000494:pharmacology
D006133:Growth Substances; D007476:Ionophores; D014667:Vasopressins; D004815:Epidermal Growth Factor; D011494:Protein Kinases; C517689:EEF2K protein, human; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054736:Elongation Factor 2 Kinase; D001920:Bradykinin; D002118:Calcium