Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells

Motomu Terasawa, Hiroshi Tokumitsu, Ryoji Kobayashi, Hiroyoshi Hidaka

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Endogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2 mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of c AMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulin-dependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-heural cells.

Original languageEnglish
Pages (from-to)417-422
Number of pages6
JournalJournal of Biochemistry
Volume110
Issue number3
Publication statusPublished - Sep 1991
Externally publishedYes

Fingerprint

Calmodulin
Protein Phosphorylation
Cytoskeleton
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Phosphorylation
Fibroblasts
Protein Kinase
Phosphopeptides
Rats
Vimentin
Proteins
Calcium-Calmodulin-Dependent Protein Kinases
KN 62
Dependent
Cell
Protein Kinases
Synapsins
Membranes
Egtazic Acid
Phosphatidylserines

ASJC Scopus subject areas

  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Physiology (medical)
  • Radiology Nuclear Medicine and imaging
  • Molecular Biology
  • Biochemistry

Cite this

Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells. / Terasawa, Motomu; Tokumitsu, Hiroshi; Kobayashi, Ryoji; Hidaka, Hiroyoshi.

In: Journal of Biochemistry, Vol. 110, No. 3, 09.1991, p. 417-422.

Research output: Contribution to journalArticle

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