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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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Dissociation of a FAK/ CAS/c-Src complex during mitosis. (a) FAK was first immunoprecipitated under condition II  from trypsinized (lane T), mitotic (lane M), and interphase  (lane I) cells, and analyzed by  immunoblotting with anti-FAK,  anti-CAS, and anti-c-Src. Phosphotyrosine levels of immunoprecipitated FAK were examined by PY20. Note that FAK  from mitotic cells showed much  reduced association with CAS or  c-Src. (b) Quantitative analyses  of the association of CAS and  c-Src with FAK, and phosphotyrosine levels of FAK immunoprecipitated from trypsinized,  mitotic, and interphase cells. The  CAS and c-Src association as  well as FAK phosphotyrosine  levels were expressed as ratios to  those of interphase FAK. Data  were obtained from five independent experiments. (c) Phosphoamino-acid analyses of FAK  immunoprecipitated from 32P-labeled, interphase, mitotic, and  trypsinized cells. PS, phosphoserine; PT, phosphothreonine;  PY, phosphotyrosine.
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Figure 3: Dissociation of a FAK/ CAS/c-Src complex during mitosis. (a) FAK was first immunoprecipitated under condition II from trypsinized (lane T), mitotic (lane M), and interphase (lane I) cells, and analyzed by immunoblotting with anti-FAK, anti-CAS, and anti-c-Src. Phosphotyrosine levels of immunoprecipitated FAK were examined by PY20. Note that FAK from mitotic cells showed much reduced association with CAS or c-Src. (b) Quantitative analyses of the association of CAS and c-Src with FAK, and phosphotyrosine levels of FAK immunoprecipitated from trypsinized, mitotic, and interphase cells. The CAS and c-Src association as well as FAK phosphotyrosine levels were expressed as ratios to those of interphase FAK. Data were obtained from five independent experiments. (c) Phosphoamino-acid analyses of FAK immunoprecipitated from 32P-labeled, interphase, mitotic, and trypsinized cells. PS, phosphoserine; PT, phosphothreonine; PY, phosphotyrosine.

Mentions: To explore the functional significance of mitosis-specific phosphorylation of FAK, we examined whether the association between FAK and FAK-associated proteins (including CAS, paxillin, c-Src, and talin) was altered. We found that the levels of CAS and c-Src associated with FAK immunoprecipitates were greatly reduced in mitotic cells (Fig. 3 a). In contrast, similar levels of paxillin or talin associated with FAK in mitotic or interphase cells were found (data not shown). Quantitative analyses (Fig. 3 b) reveal the levels of CAS or c-Src associated with mitotic FAK are 16 ± 9% and 13 ± 10% of those associated with interphase FAK. The difference is not due to the rounded morphology of mitotic cells because FAK immunoprecipitated from rounded trypsinized cells was associated with considerable amounts of CAS and c-Src. The levels of CAS and c-Src were 83 ± 23% and 70 ± 34% of the interphase levels, respectively. The dissociation of FAK/CAS/ c-Src complex in mitotic cells is not due to nocodazole treatment because similar dissociation was observed with mitotic cells prepared without nocodazole treatment.


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)

Dissociation of a FAK/ CAS/c-Src complex during mitosis. (a) FAK was first immunoprecipitated under condition II  from trypsinized (lane T), mitotic (lane M), and interphase  (lane I) cells, and analyzed by  immunoblotting with anti-FAK,  anti-CAS, and anti-c-Src. Phosphotyrosine levels of immunoprecipitated FAK were examined by PY20. Note that FAK  from mitotic cells showed much  reduced association with CAS or  c-Src. (b) Quantitative analyses  of the association of CAS and  c-Src with FAK, and phosphotyrosine levels of FAK immunoprecipitated from trypsinized,  mitotic, and interphase cells. The  CAS and c-Src association as  well as FAK phosphotyrosine  levels were expressed as ratios to  those of interphase FAK. Data  were obtained from five independent experiments. (c) Phosphoamino-acid analyses of FAK  immunoprecipitated from 32P-labeled, interphase, mitotic, and  trypsinized cells. PS, phosphoserine; PT, phosphothreonine;  PY, phosphotyrosine.
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Figure 3: Dissociation of a FAK/ CAS/c-Src complex during mitosis. (a) FAK was first immunoprecipitated under condition II from trypsinized (lane T), mitotic (lane M), and interphase (lane I) cells, and analyzed by immunoblotting with anti-FAK, anti-CAS, and anti-c-Src. Phosphotyrosine levels of immunoprecipitated FAK were examined by PY20. Note that FAK from mitotic cells showed much reduced association with CAS or c-Src. (b) Quantitative analyses of the association of CAS and c-Src with FAK, and phosphotyrosine levels of FAK immunoprecipitated from trypsinized, mitotic, and interphase cells. The CAS and c-Src association as well as FAK phosphotyrosine levels were expressed as ratios to those of interphase FAK. Data were obtained from five independent experiments. (c) Phosphoamino-acid analyses of FAK immunoprecipitated from 32P-labeled, interphase, mitotic, and trypsinized cells. PS, phosphoserine; PT, phosphothreonine; PY, phosphotyrosine.
Mentions: To explore the functional significance of mitosis-specific phosphorylation of FAK, we examined whether the association between FAK and FAK-associated proteins (including CAS, paxillin, c-Src, and talin) was altered. We found that the levels of CAS and c-Src associated with FAK immunoprecipitates were greatly reduced in mitotic cells (Fig. 3 a). In contrast, similar levels of paxillin or talin associated with FAK in mitotic or interphase cells were found (data not shown). Quantitative analyses (Fig. 3 b) reveal the levels of CAS or c-Src associated with mitotic FAK are 16 ± 9% and 13 ± 10% of those associated with interphase FAK. The difference is not due to the rounded morphology of mitotic cells because FAK immunoprecipitated from rounded trypsinized cells was associated with considerable amounts of CAS and c-Src. The levels of CAS and c-Src were 83 ± 23% and 70 ± 34% of the interphase levels, respectively. The dissociation of FAK/CAS/ c-Src complex in mitotic cells is not due to nocodazole treatment because similar dissociation was observed with mitotic cells prepared without nocodazole treatment.

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