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Dissociation of FAK/p130(CAS)/c-Src complex during mitosis: role of mitosis-specific serine phosphorylation of FAK.

Yamakita Y, Totsukawa G, Yamashiro S, Fry D, Zhang X, Hanks SK, Matsumura F - J. Cell Biol. (1999)

Bottom Line: We have found two significant alterations in FAK-mediated signal transduction during mitosis.Second, mitotic FAK shows decreased binding to a peptide mimicking the cytoplasmic domain of beta-integrin when compared with FAK of interphase cells.These results suggest that mitosis-specific modification of FAK uncouples signal transduction pathways involving integrin, CAS, and c-Src, and may maintain FAK in an inactive state until post-mitotic spreading.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Rutgers University, Nelson Labs, Piscataway, New Jersey 08855, USA.

ABSTRACT
At mitosis, focal adhesions disassemble and the signal transduction from focal adhesions is inactivated. We have found that components of focal adhesions including focal adhesion kinase (FAK), paxillin, and p130(CAS) (CAS) are serine/threonine phosphorylated during mitosis when all three proteins are tyrosine dephosphorylated. Mitosis-specific phosphorylation continues past cytokinesis and is reversed during post-mitotic cell spreading. We have found two significant alterations in FAK-mediated signal transduction during mitosis. First, the association of FAK with CAS or c-Src is greatly inhibited, with levels decreasing to 16 and 13% of the interphase levels, respectively. Second, mitotic FAK shows decreased binding to a peptide mimicking the cytoplasmic domain of beta-integrin when compared with FAK of interphase cells. Mitosis-specific phosphorylation is responsible for the disruption of FAK/CAS binding because dephosphorylation of mitotic FAK in vitro by protein serine/threonine phosphatase 1 restores the ability of FAK to associate with CAS, though not with c-Src. These results suggest that mitosis-specific modification of FAK uncouples signal transduction pathways involving integrin, CAS, and c-Src, and may maintain FAK in an inactive state until post-mitotic spreading.

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Reconstitution of a  FAK/CAS complex following  dephosphorylation of mitotic  FAK. (a) FAK was immunoprecipitated under condition I from  mitotic cells and divided into  three equal parts for further  treatment: lane 1, neither incubated with interphase extracts  nor treated with PP1; lane 2, incubated with interphase extracts  following treatment with PP1;  lane 3, incubated with interphase  extracts. After extensive washing, the association of FAK with  CAS was examined by blotting  with the anti-FAK antibody or  anti-CAS antibody. Phosphotyrosine levels of FAK were examined by PY20. For comparison,  FAK immunoprecipitates from  interphase cells were blotted with the same antibodies (lane 4). (b) Quantitative analyses. The CAS reassociation and phosphotyrosine  levels of FAK are expressed as ratios to those found in FAK incubated with interphase extracts but without prior PP1 treatment  (−PP1). Data were obtained from five independent experiments.
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Figure 4: Reconstitution of a FAK/CAS complex following dephosphorylation of mitotic FAK. (a) FAK was immunoprecipitated under condition I from mitotic cells and divided into three equal parts for further treatment: lane 1, neither incubated with interphase extracts nor treated with PP1; lane 2, incubated with interphase extracts following treatment with PP1; lane 3, incubated with interphase extracts. After extensive washing, the association of FAK with CAS was examined by blotting with the anti-FAK antibody or anti-CAS antibody. Phosphotyrosine levels of FAK were examined by PY20. For comparison, FAK immunoprecipitates from interphase cells were blotted with the same antibodies (lane 4). (b) Quantitative analyses. The CAS reassociation and phosphotyrosine levels of FAK are expressed as ratios to those found in FAK incubated with interphase extracts but without prior PP1 treatment (−PP1). Data were obtained from five independent experiments.

Mentions: To test whether mitosis-specific serine phosphorylation or tyrosine dephosphorylation causes the disruption of the FAK/CAS/c-Src complex, we dephosphorylated mitotic FAK by PP1, and examined whether serine dephosphorylated FAK was able to reassociate with CAS or c-Src. FAK was first immunoprecipitated from mitotic cells under condition I. One-third of the immunoprecipitate was used to confirm that neither CAS nor c-Src is associated with mitotic FAK (lane 1 of Fig. 4 a). The rest of the immunoprecipitate was divided in half; one (lane 2) was dephosphorylated by PP1, and the other (lane 3) was untreated. Fig. 4 a shows PP1 treatment resulted in the loss of mobility shift of FAK, confirming dephosphorylation of mitotic FAK. Both PP1 treated and untreated FAK were incubated with interphase extracts. After extensive washing, the binding of CAS or c-Src was examined by immunoblotting. Dephosphorylated FAK (Fig. 4 a, lane 2) was able to reassociate with CAS of interphase extracts (second panel). The level of CAS reassociation was comparable to interphase FAK immunoprecipitates (lane 4). In contrast, the phosphatase untreated mitotic FAK (lane 3) showed reduced binding to CAS. Quantitative analyses revealed that the binding of CAS to dephosphorylated FAK is more than three times higher than for untreated FAK (Fig. 4 b).


Dissociation of FAK/p130(CAS)/c-Src complex during mitosis: role of mitosis-specific serine phosphorylation of FAK.

Yamakita Y, Totsukawa G, Yamashiro S, Fry D, Zhang X, Hanks SK, Matsumura F - J. Cell Biol. (1999)

Reconstitution of a  FAK/CAS complex following  dephosphorylation of mitotic  FAK. (a) FAK was immunoprecipitated under condition I from  mitotic cells and divided into  three equal parts for further  treatment: lane 1, neither incubated with interphase extracts  nor treated with PP1; lane 2, incubated with interphase extracts  following treatment with PP1;  lane 3, incubated with interphase  extracts. After extensive washing, the association of FAK with  CAS was examined by blotting  with the anti-FAK antibody or  anti-CAS antibody. Phosphotyrosine levels of FAK were examined by PY20. For comparison,  FAK immunoprecipitates from  interphase cells were blotted with the same antibodies (lane 4). (b) Quantitative analyses. The CAS reassociation and phosphotyrosine  levels of FAK are expressed as ratios to those found in FAK incubated with interphase extracts but without prior PP1 treatment  (−PP1). Data were obtained from five independent experiments.
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Related In: Results  -  Collection

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Figure 4: Reconstitution of a FAK/CAS complex following dephosphorylation of mitotic FAK. (a) FAK was immunoprecipitated under condition I from mitotic cells and divided into three equal parts for further treatment: lane 1, neither incubated with interphase extracts nor treated with PP1; lane 2, incubated with interphase extracts following treatment with PP1; lane 3, incubated with interphase extracts. After extensive washing, the association of FAK with CAS was examined by blotting with the anti-FAK antibody or anti-CAS antibody. Phosphotyrosine levels of FAK were examined by PY20. For comparison, FAK immunoprecipitates from interphase cells were blotted with the same antibodies (lane 4). (b) Quantitative analyses. The CAS reassociation and phosphotyrosine levels of FAK are expressed as ratios to those found in FAK incubated with interphase extracts but without prior PP1 treatment (−PP1). Data were obtained from five independent experiments.
Mentions: To test whether mitosis-specific serine phosphorylation or tyrosine dephosphorylation causes the disruption of the FAK/CAS/c-Src complex, we dephosphorylated mitotic FAK by PP1, and examined whether serine dephosphorylated FAK was able to reassociate with CAS or c-Src. FAK was first immunoprecipitated from mitotic cells under condition I. One-third of the immunoprecipitate was used to confirm that neither CAS nor c-Src is associated with mitotic FAK (lane 1 of Fig. 4 a). The rest of the immunoprecipitate was divided in half; one (lane 2) was dephosphorylated by PP1, and the other (lane 3) was untreated. Fig. 4 a shows PP1 treatment resulted in the loss of mobility shift of FAK, confirming dephosphorylation of mitotic FAK. Both PP1 treated and untreated FAK were incubated with interphase extracts. After extensive washing, the binding of CAS or c-Src was examined by immunoblotting. Dephosphorylated FAK (Fig. 4 a, lane 2) was able to reassociate with CAS of interphase extracts (second panel). The level of CAS reassociation was comparable to interphase FAK immunoprecipitates (lane 4). In contrast, the phosphatase untreated mitotic FAK (lane 3) showed reduced binding to CAS. Quantitative analyses revealed that the binding of CAS to dephosphorylated FAK is more than three times higher than for untreated FAK (Fig. 4 b).

Bottom Line: We have found two significant alterations in FAK-mediated signal transduction during mitosis.Second, mitotic FAK shows decreased binding to a peptide mimicking the cytoplasmic domain of beta-integrin when compared with FAK of interphase cells.These results suggest that mitosis-specific modification of FAK uncouples signal transduction pathways involving integrin, CAS, and c-Src, and may maintain FAK in an inactive state until post-mitotic spreading.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Rutgers University, Nelson Labs, Piscataway, New Jersey 08855, USA.

ABSTRACT
At mitosis, focal adhesions disassemble and the signal transduction from focal adhesions is inactivated. We have found that components of focal adhesions including focal adhesion kinase (FAK), paxillin, and p130(CAS) (CAS) are serine/threonine phosphorylated during mitosis when all three proteins are tyrosine dephosphorylated. Mitosis-specific phosphorylation continues past cytokinesis and is reversed during post-mitotic cell spreading. We have found two significant alterations in FAK-mediated signal transduction during mitosis. First, the association of FAK with CAS or c-Src is greatly inhibited, with levels decreasing to 16 and 13% of the interphase levels, respectively. Second, mitotic FAK shows decreased binding to a peptide mimicking the cytoplasmic domain of beta-integrin when compared with FAK of interphase cells. Mitosis-specific phosphorylation is responsible for the disruption of FAK/CAS binding because dephosphorylation of mitotic FAK in vitro by protein serine/threonine phosphatase 1 restores the ability of FAK to associate with CAS, though not with c-Src. These results suggest that mitosis-specific modification of FAK uncouples signal transduction pathways involving integrin, CAS, and c-Src, and may maintain FAK in an inactive state until post-mitotic spreading.

Show MeSH
Related in: MedlinePlus