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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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Serine phosphorylation is responsible for the mobility  shifts. (a) Loss of mobility shifts by the treatment with PP1. FAK,  CAS, and paxillin (PAX) were immunoprecipitated under condition I from mitotic (odd numbered lanes) or from interphase  (even numbered lanes) cells, and half of the immunoprecipitates  (lanes 3, 4, 7, 8, 11, and 12) were treated with PP1 to dephosphorylate phosphoserine and phosphothreonine. Note that PP1  treatment eliminates or greatly decreases the mobility shifts  shown by mitotic FAK (compare lanes 1 and 3), CAS (compare  lanes 5 and 7), and paxillin (compare lanes 9 and 11). (b) Tyrosine dephosphorylation of FAK, CAS, and paxillin during mitosis. FAK, CAS, and paxillin (PAX) were again immunoprecipitated under condition I from mitotic (lanes M) and interphase  (lanes I) cells. The immunoprecipitates were first immunoblotted  with PY20 (top; lanes 1, 2, 5, 6, 9, and 10), and then reprobed with  FAK, CAS, and paxillin antibodies (bottom; lanes 3, 4, 7, 8, 11,  and 12). Note that PY20 reacts strongly with the immunoprecipitates prepared from interphase but not from mitotic cells, indicating that mitotic FAK, CAS, and paxillin are dephosphorylated at  tyrosine residues. (c) Phosphate incorporation of FAK, CAS, and  paxillin. FAK, CAS, and paxillin were immunoprecipitated under condition I from mitotic (lanes M) and interphase (lanes I)  cells that had been labeled in vivo with 32P inorganic phosphate.  The 32P-labeled immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. The levels of 32P incorporation in mitotic FAK, CAS, and paxillin are not greatly increased  because serine phosphorylation is negated by tyrosine dephosphorylation.
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Figure 2: Serine phosphorylation is responsible for the mobility shifts. (a) Loss of mobility shifts by the treatment with PP1. FAK, CAS, and paxillin (PAX) were immunoprecipitated under condition I from mitotic (odd numbered lanes) or from interphase (even numbered lanes) cells, and half of the immunoprecipitates (lanes 3, 4, 7, 8, 11, and 12) were treated with PP1 to dephosphorylate phosphoserine and phosphothreonine. Note that PP1 treatment eliminates or greatly decreases the mobility shifts shown by mitotic FAK (compare lanes 1 and 3), CAS (compare lanes 5 and 7), and paxillin (compare lanes 9 and 11). (b) Tyrosine dephosphorylation of FAK, CAS, and paxillin during mitosis. FAK, CAS, and paxillin (PAX) were again immunoprecipitated under condition I from mitotic (lanes M) and interphase (lanes I) cells. The immunoprecipitates were first immunoblotted with PY20 (top; lanes 1, 2, 5, 6, 9, and 10), and then reprobed with FAK, CAS, and paxillin antibodies (bottom; lanes 3, 4, 7, 8, 11, and 12). Note that PY20 reacts strongly with the immunoprecipitates prepared from interphase but not from mitotic cells, indicating that mitotic FAK, CAS, and paxillin are dephosphorylated at tyrosine residues. (c) Phosphate incorporation of FAK, CAS, and paxillin. FAK, CAS, and paxillin were immunoprecipitated under condition I from mitotic (lanes M) and interphase (lanes I) cells that had been labeled in vivo with 32P inorganic phosphate. The 32P-labeled immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. The levels of 32P incorporation in mitotic FAK, CAS, and paxillin are not greatly increased because serine phosphorylation is negated by tyrosine dephosphorylation.

Mentions: To test whether the mitotic modification is serine/threonine phosphorylation, we examined the effect of phosphatase treatment on the mobility shifts. FAK, CAS, and paxillin were again immunoprecipitated from mitotic and interphase cells. One-half of each immunoprecipitate was treated with serine/threonine protein phosphatase 1 (PP1), and immunoblotted with specific antibodies. As Fig. 2 a shows, PP1 treatment eliminated or greatly decreased the mobility shifts shown by mitotic FAK, CAS, and paxillin, indicating that serine/threonine phosphorylation is largely responsible for the mobility shifts.


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)

Serine phosphorylation is responsible for the mobility  shifts. (a) Loss of mobility shifts by the treatment with PP1. FAK,  CAS, and paxillin (PAX) were immunoprecipitated under condition I from mitotic (odd numbered lanes) or from interphase  (even numbered lanes) cells, and half of the immunoprecipitates  (lanes 3, 4, 7, 8, 11, and 12) were treated with PP1 to dephosphorylate phosphoserine and phosphothreonine. Note that PP1  treatment eliminates or greatly decreases the mobility shifts  shown by mitotic FAK (compare lanes 1 and 3), CAS (compare  lanes 5 and 7), and paxillin (compare lanes 9 and 11). (b) Tyrosine dephosphorylation of FAK, CAS, and paxillin during mitosis. FAK, CAS, and paxillin (PAX) were again immunoprecipitated under condition I from mitotic (lanes M) and interphase  (lanes I) cells. The immunoprecipitates were first immunoblotted  with PY20 (top; lanes 1, 2, 5, 6, 9, and 10), and then reprobed with  FAK, CAS, and paxillin antibodies (bottom; lanes 3, 4, 7, 8, 11,  and 12). Note that PY20 reacts strongly with the immunoprecipitates prepared from interphase but not from mitotic cells, indicating that mitotic FAK, CAS, and paxillin are dephosphorylated at  tyrosine residues. (c) Phosphate incorporation of FAK, CAS, and  paxillin. FAK, CAS, and paxillin were immunoprecipitated under condition I from mitotic (lanes M) and interphase (lanes I)  cells that had been labeled in vivo with 32P inorganic phosphate.  The 32P-labeled immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. The levels of 32P incorporation in mitotic FAK, CAS, and paxillin are not greatly increased  because serine phosphorylation is negated by tyrosine dephosphorylation.
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Related In: Results  -  Collection

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Figure 2: Serine phosphorylation is responsible for the mobility shifts. (a) Loss of mobility shifts by the treatment with PP1. FAK, CAS, and paxillin (PAX) were immunoprecipitated under condition I from mitotic (odd numbered lanes) or from interphase (even numbered lanes) cells, and half of the immunoprecipitates (lanes 3, 4, 7, 8, 11, and 12) were treated with PP1 to dephosphorylate phosphoserine and phosphothreonine. Note that PP1 treatment eliminates or greatly decreases the mobility shifts shown by mitotic FAK (compare lanes 1 and 3), CAS (compare lanes 5 and 7), and paxillin (compare lanes 9 and 11). (b) Tyrosine dephosphorylation of FAK, CAS, and paxillin during mitosis. FAK, CAS, and paxillin (PAX) were again immunoprecipitated under condition I from mitotic (lanes M) and interphase (lanes I) cells. The immunoprecipitates were first immunoblotted with PY20 (top; lanes 1, 2, 5, 6, 9, and 10), and then reprobed with FAK, CAS, and paxillin antibodies (bottom; lanes 3, 4, 7, 8, 11, and 12). Note that PY20 reacts strongly with the immunoprecipitates prepared from interphase but not from mitotic cells, indicating that mitotic FAK, CAS, and paxillin are dephosphorylated at tyrosine residues. (c) Phosphate incorporation of FAK, CAS, and paxillin. FAK, CAS, and paxillin were immunoprecipitated under condition I from mitotic (lanes M) and interphase (lanes I) cells that had been labeled in vivo with 32P inorganic phosphate. The 32P-labeled immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. The levels of 32P incorporation in mitotic FAK, CAS, and paxillin are not greatly increased because serine phosphorylation is negated by tyrosine dephosphorylation.
Mentions: To test whether the mitotic modification is serine/threonine phosphorylation, we examined the effect of phosphatase treatment on the mobility shifts. FAK, CAS, and paxillin were again immunoprecipitated from mitotic and interphase cells. One-half of each immunoprecipitate was treated with serine/threonine protein phosphatase 1 (PP1), and immunoblotted with specific antibodies. As Fig. 2 a shows, PP1 treatment eliminated or greatly decreased the mobility shifts shown by mitotic FAK, CAS, and paxillin, indicating that serine/threonine phosphorylation is largely responsible for the mobility shifts.

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