Rapid activation of calmodulin-dependent protein kinase III in mitogen-stimulated human fibroblasts. Correlation with intracellular Ca2+ transients

H. C. Palfrey, A. C. Nairn, Leslie Muldoon, M. L. Villereal

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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 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 languageEnglish (US)
Pages (from-to)9785-9792
Number of pages8
JournalJournal of Biological Chemistry
Volume262
Issue number20
StatePublished - 1987
Externally publishedYes

Fingerprint

Elongation Factor 2 Kinase
Calmodulin
Fibroblasts
Mitogens
Phosphorylation
Chemical activation
Bradykinin
Serum
Vasopressins
Epidermal Growth Factor
Intercellular Signaling Peptides and Proteins
Cells
Phosphopeptides
Time and motion study
Peptide Mapping
Fura-2
Phosphoproteins
Ionophores
Calcimycin
Threonine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Rapid activation of calmodulin-dependent protein kinase III in mitogen-stimulated human fibroblasts. Correlation with intracellular Ca2+ transients. / Palfrey, H. C.; Nairn, A. C.; Muldoon, Leslie; Villereal, M. L.

In: Journal of Biological Chemistry, Vol. 262, No. 20, 1987, p. 9785-9792.

Research output: Contribution to journalArticle

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T1 - Rapid activation of calmodulin-dependent protein kinase III in mitogen-stimulated human fibroblasts. Correlation with intracellular Ca2+ transients

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AU - Nairn, A. C.

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AU - Villereal, M. L.

PY - 1987

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N2 - 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.

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