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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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Tyrosine rephosphorylation of FAK (a), paxillin (b),  and CAS (c) during post-mitotic cell spreading. FAK, CAS, and  paxillin were immunoprecipitated from interphase cells (I), mitotic cells (M), and cells released from mitotic arrest (numbered  lanes, n = min removed from nocodazole). The immunoprecipitates were transferred to PVDF membranes, first immunoblotted  with PY20, then reprobed with the antibodies against FAK, paxillin, and CAS. The levels of phosphotyrosine are shown by ratios  (100% for interphase level) of the levels of PY20 reactivities divided by the levels of FAK, paxillin, or CAS. Note that FAK exhibits the fastest recovery of tyrosine rephosphorylation as well  as the greatest increase in tyrosine phosphorylation during 80– 180 min after the release of mitotic arrest.
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Figure 7: Tyrosine rephosphorylation of FAK (a), paxillin (b), and CAS (c) during post-mitotic cell spreading. FAK, CAS, and paxillin were immunoprecipitated from interphase cells (I), mitotic cells (M), and cells released from mitotic arrest (numbered lanes, n = min removed from nocodazole). The immunoprecipitates were transferred to PVDF membranes, first immunoblotted with PY20, then reprobed with the antibodies against FAK, paxillin, and CAS. The levels of phosphotyrosine are shown by ratios (100% for interphase level) of the levels of PY20 reactivities divided by the levels of FAK, paxillin, or CAS. Note that FAK exhibits the fastest recovery of tyrosine rephosphorylation as well as the greatest increase in tyrosine phosphorylation during 80– 180 min after the release of mitotic arrest.

Mentions: Because tyrosine phosphorylation plays an important role in signal transduction and organization of focal adhesions, the time course of tyrosine rephosphorylation was also examined. Immunoprecipitates of FAK, paxillin, and CAS prepared at different stages of cell cycle were blotted with PY20, and reprobed with the antibodies against each protein for normalization. As Fig. 7 shows, the proteins were tyrosine rephosphorylated between 80 and 180 min after release of mitotic arrest (corresponding to post- mitotic cell spreading). FAK exhibited the fastest kinetics of tyrosine rephosphorylation and the largest increase in tyrosine phosphorylation at 180 min. The PY20 reactivity of FAK was increased threefold between 80 and 180 min, while paxillin gradually increased only 134%. CAS showed the slowest tyrosine rephosphorylation, the PY20 reactivity of CAS was <15% of the interphase level, even at 120 min. In the next 1 h, however, the PY20 reactivity of CAS increased to 200%.


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)

Tyrosine rephosphorylation of FAK (a), paxillin (b),  and CAS (c) during post-mitotic cell spreading. FAK, CAS, and  paxillin were immunoprecipitated from interphase cells (I), mitotic cells (M), and cells released from mitotic arrest (numbered  lanes, n = min removed from nocodazole). The immunoprecipitates were transferred to PVDF membranes, first immunoblotted  with PY20, then reprobed with the antibodies against FAK, paxillin, and CAS. The levels of phosphotyrosine are shown by ratios  (100% for interphase level) of the levels of PY20 reactivities divided by the levels of FAK, paxillin, or CAS. Note that FAK exhibits the fastest recovery of tyrosine rephosphorylation as well  as the greatest increase in tyrosine phosphorylation during 80– 180 min after the release of mitotic arrest.
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Related In: Results  -  Collection

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Figure 7: Tyrosine rephosphorylation of FAK (a), paxillin (b), and CAS (c) during post-mitotic cell spreading. FAK, CAS, and paxillin were immunoprecipitated from interphase cells (I), mitotic cells (M), and cells released from mitotic arrest (numbered lanes, n = min removed from nocodazole). The immunoprecipitates were transferred to PVDF membranes, first immunoblotted with PY20, then reprobed with the antibodies against FAK, paxillin, and CAS. The levels of phosphotyrosine are shown by ratios (100% for interphase level) of the levels of PY20 reactivities divided by the levels of FAK, paxillin, or CAS. Note that FAK exhibits the fastest recovery of tyrosine rephosphorylation as well as the greatest increase in tyrosine phosphorylation during 80– 180 min after the release of mitotic arrest.
Mentions: Because tyrosine phosphorylation plays an important role in signal transduction and organization of focal adhesions, the time course of tyrosine rephosphorylation was also examined. Immunoprecipitates of FAK, paxillin, and CAS prepared at different stages of cell cycle were blotted with PY20, and reprobed with the antibodies against each protein for normalization. As Fig. 7 shows, the proteins were tyrosine rephosphorylated between 80 and 180 min after release of mitotic arrest (corresponding to post- mitotic cell spreading). FAK exhibited the fastest kinetics of tyrosine rephosphorylation and the largest increase in tyrosine phosphorylation at 180 min. The PY20 reactivity of FAK was increased threefold between 80 and 180 min, while paxillin gradually increased only 134%. CAS showed the slowest tyrosine rephosphorylation, the PY20 reactivity of CAS was <15% of the interphase level, even at 120 min. In the next 1 h, however, the PY20 reactivity of CAS increased to 200%.

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