The functional role of CrkII in actin cytoskeleton organization and mitogenesis

Naoki Nakashima, David W. Rose, Sen Xiao, Katsuya Egawa, Stuart S. Martin, Tetsuro Haruta, Alan R. Saltiel, Jerrold M. Olefsky

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Abstract

Crk is a member of a family of adapter proteins predominantly composed of Src homology 2 and 3 domains, whose role in signaling pathways is presently unclear. Using an in situ electroporation system which permits the introduction of glutathione S-transferase (GST) fusion proteins into cells, we found that c-CrkII bound to p130(cas), but not to paxillin in serum- starved rat-1 fibroblasts overexpressing the human insulin receptor (HIRc cells) in vivo. 17 nM insulin stimulation dissociated the binding of c-CrkII to p130(cas), whereas 13 nM insulin-like growth factor-I, 16 nM epidermal growth factor (EGF), and 10% serum each showed little or no effect. We found that stress fiber formation is consistent with a change in the p130(cas)·c- CrkII interactions before and after growth factor stimulation. Microinjection of either GST-CrkSH2 or -Crk-(N)SH3 domains, or anti-Crk antibody each inhibited stress fiber formation before and after insulin-like growth factor- I, EGF, and serum stimulation. Insulin stimulation by itself caused stress fiber breakdown and there was no additive effect of microinjection. Microinjection of anti-p130(cas) antibody also blocked stress fiber formation in quiescent cells. Microinjection of the Crk-inhibitory reagents also inhibited DNA synthesis after insulin-like growth factor-I, EGF, and serum stimulation, but not after insulin. These data suggest that the complex containing p130(cas)·c-CrkII may play a crucial role in actin cytoskeleton organization and in anchorage-dependent DNA synthesis.

Original languageEnglish
Pages (from-to)3001-3008
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number5
DOIs
Publication statusPublished - Jan 29 1999

Fingerprint

Stress Fibers
Microinjections
Actin Cytoskeleton
Actins
Insulin-Like Growth Factor I
Epidermal Growth Factor
Organizations
Fibers
Insulin
Glutathione Transferase
Serum
Paxillin
Electroporation
src Homology Domains
Antibodies
DNA
Fibroblasts
Rats
Anti-Idiotypic Antibodies
Intercellular Signaling Peptides and Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Nakashima, N., Rose, D. W., Xiao, S., Egawa, K., Martin, S. S., Haruta, T., ... Olefsky, J. M. (1999). The functional role of CrkII in actin cytoskeleton organization and mitogenesis. Journal of Biological Chemistry, 274(5), 3001-3008. https://doi.org/10.1074/jbc.274.5.3001

The functional role of CrkII in actin cytoskeleton organization and mitogenesis. / Nakashima, Naoki; Rose, David W.; Xiao, Sen; Egawa, Katsuya; Martin, Stuart S.; Haruta, Tetsuro; Saltiel, Alan R.; Olefsky, Jerrold M.

In: Journal of Biological Chemistry, Vol. 274, No. 5, 29.01.1999, p. 3001-3008.

Research output: Contribution to journalArticle

Nakashima, N, Rose, DW, Xiao, S, Egawa, K, Martin, SS, Haruta, T, Saltiel, AR & Olefsky, JM 1999, 'The functional role of CrkII in actin cytoskeleton organization and mitogenesis', Journal of Biological Chemistry, vol. 274, no. 5, pp. 3001-3008. https://doi.org/10.1074/jbc.274.5.3001
Nakashima, Naoki ; Rose, David W. ; Xiao, Sen ; Egawa, Katsuya ; Martin, Stuart S. ; Haruta, Tetsuro ; Saltiel, Alan R. ; Olefsky, Jerrold M. / The functional role of CrkII in actin cytoskeleton organization and mitogenesis. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 5. pp. 3001-3008.
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