Abstract
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.
Highlights
From the Departmentof Pharmacological and Physiological Sciences, University of Chicago, Chicago,Illinois 60637 and the §Department of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York10021
Stimulation of 32P-la- Mitogeneffects on phosphatidylinositol 4,5-bisphosphate beled serum-deprived fibroblasts with serum, individ- turnover lead to the generation of diacylglycerol within ual growth factors,or Ca2+ ionophores resulted in enzyme protein kinase C (PK-C) [4]
These results implied the presence of CaM-PK I11 in HSWP cells because it has been previously shown that this enzyme is the only purified Ca2+-sensitiveprotein kinase capable of phosphorylating the M, 100,000substrate in vitro [11].In addition, we were able to demonstrate the presence of CaM-PK I and I1 in both types of fibroblast using the exogenous substrate Synapsin I.'
Summary
Ca2+-regulatedPhosphorylation in Fibroblast Extracts-Cytoplasmic extracts were prepared and tested for their ability to phosphorylate endogenous substrates. 9 and 11)and WI38 proteins and M,100,000 from other tissues; (ii)the major phosphorylated amino acid was threonine (see Fig. 4 0 ) ; (iii) the protein could be recognized on Western blots by a polyclonal antibody raised against the pancreatic M, 100,000 protein (not shown) and could be immunoprecipitated from cell extracts by the same antibody (see below) These results implied the presence of CaM-PK I11 in HSWP cells because it has been previously shown that this enzyme is the only purified Ca2+-sensitiveprotein kinase capable of phosphorylating the M, 100,000substrate in vitro [11].In addition, we were able to demonstrate the presence of CaM-PK I and I1 in both types of fibroblast using the exogenous substrate Synapsin I.'.
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