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 protien 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 M(r) 100,000 and pI 6.8 (M(r) 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 Plafrey, 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 M(r) 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 M(r) 100,000 closely paralleled but lagged slightly behind the Ca2+ transient (measured with fura-2). Thus, dephosphorylation of M(r) 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 M(r) 100,000 were independent of external Ca2+, whereas the effects of epidermal growth factor and A23187 required external Ca2+. Phosphorylation of M(r) 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.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1987|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology