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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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Reduced binding of  mitotic FAK to a peptide mimicking the cytoplasmic domain of  integrin beta subunit. (a) Total  cell lysates were prepared from  trypsinized (lane T), mitotic  (lane M), and interphase (lane I)  cells, and were immunoblotted  with the anti-FAK antibody to  confirm that they contain approximately equal amounts of  FAK. SP-1 peptide–conjugated  Sepharose beads were incubated  with diluted (9×) extracts of  trypsinized (lane T), mitotic  (lane M), and interphase (lane I)  cells. After extensive washing,  bound fractions were analyzed  by SDS-PAGE, followed by immunoblotting with the anti-FAK  antibody. (b) Quantitative analyses of SP1 binding. The extracts from trypsinized (T), mitotic (M), and interphase (I) were serially diluted as indicated and bound FAK  was determined by densitometry of immunoblots using the FAK antibody. The levels of bound FAK were expressed as ratios to the  level of bound FAK after incubation with 9× diluted interphase extracts.
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Figure 5: Reduced binding of mitotic FAK to a peptide mimicking the cytoplasmic domain of integrin beta subunit. (a) Total cell lysates were prepared from trypsinized (lane T), mitotic (lane M), and interphase (lane I) cells, and were immunoblotted with the anti-FAK antibody to confirm that they contain approximately equal amounts of FAK. SP-1 peptide–conjugated Sepharose beads were incubated with diluted (9×) extracts of trypsinized (lane T), mitotic (lane M), and interphase (lane I) cells. After extensive washing, bound fractions were analyzed by SDS-PAGE, followed by immunoblotting with the anti-FAK antibody. (b) Quantitative analyses of SP1 binding. The extracts from trypsinized (T), mitotic (M), and interphase (I) were serially diluted as indicated and bound FAK was determined by densitometry of immunoblots using the FAK antibody. The levels of bound FAK were expressed as ratios to the level of bound FAK after incubation with 9× diluted interphase extracts.

Mentions: Both FAK and paxillin have been reported to bind directly to a peptide (SP1) representing a membrane-proximal region of the cytoplasmic domain of the beta subunit of integrin (Schaller et al., 1995). We examined whether mitotic FAK or paxillin exhibited altered binding to the SP1 peptide. SP1 peptide–conjugated beads were incubated in varying concentrations of extracts prepared from mitotic, interphase, and trypsinized cells. After washing, levels of bound FAK and paxillin were detected by immunoblotting. As Fig. 5 a shows, mitotic FAK bound to SP1 peptide–conjugated beads less (lane M) than FAK from interphase (lane I), or trypsinized (lane T) cells. Quantitative analysis (Fig. 5 b) revealed the binding of mitotic FAK is 24–33% of the level shown by interphase FAK, while FAK from trypsinized cells showed 65–85% binding of the interphase level (Fig. 5 b). The binding of paxillin to SP1 beads, in contrast, revealed no difference between mitotic and interphase cells (data not shown).


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)

Reduced binding of  mitotic FAK to a peptide mimicking the cytoplasmic domain of  integrin beta subunit. (a) Total  cell lysates were prepared from  trypsinized (lane T), mitotic  (lane M), and interphase (lane I)  cells, and were immunoblotted  with the anti-FAK antibody to  confirm that they contain approximately equal amounts of  FAK. SP-1 peptide–conjugated  Sepharose beads were incubated  with diluted (9×) extracts of  trypsinized (lane T), mitotic  (lane M), and interphase (lane I)  cells. After extensive washing,  bound fractions were analyzed  by SDS-PAGE, followed by immunoblotting with the anti-FAK  antibody. (b) Quantitative analyses of SP1 binding. The extracts from trypsinized (T), mitotic (M), and interphase (I) were serially diluted as indicated and bound FAK  was determined by densitometry of immunoblots using the FAK antibody. The levels of bound FAK were expressed as ratios to the  level of bound FAK after incubation with 9× diluted interphase extracts.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132894&req=5

Figure 5: Reduced binding of mitotic FAK to a peptide mimicking the cytoplasmic domain of integrin beta subunit. (a) Total cell lysates were prepared from trypsinized (lane T), mitotic (lane M), and interphase (lane I) cells, and were immunoblotted with the anti-FAK antibody to confirm that they contain approximately equal amounts of FAK. SP-1 peptide–conjugated Sepharose beads were incubated with diluted (9×) extracts of trypsinized (lane T), mitotic (lane M), and interphase (lane I) cells. After extensive washing, bound fractions were analyzed by SDS-PAGE, followed by immunoblotting with the anti-FAK antibody. (b) Quantitative analyses of SP1 binding. The extracts from trypsinized (T), mitotic (M), and interphase (I) were serially diluted as indicated and bound FAK was determined by densitometry of immunoblots using the FAK antibody. The levels of bound FAK were expressed as ratios to the level of bound FAK after incubation with 9× diluted interphase extracts.
Mentions: Both FAK and paxillin have been reported to bind directly to a peptide (SP1) representing a membrane-proximal region of the cytoplasmic domain of the beta subunit of integrin (Schaller et al., 1995). We examined whether mitotic FAK or paxillin exhibited altered binding to the SP1 peptide. SP1 peptide–conjugated beads were incubated in varying concentrations of extracts prepared from mitotic, interphase, and trypsinized cells. After washing, levels of bound FAK and paxillin were detected by immunoblotting. As Fig. 5 a shows, mitotic FAK bound to SP1 peptide–conjugated beads less (lane M) than FAK from interphase (lane I), or trypsinized (lane T) cells. Quantitative analysis (Fig. 5 b) revealed the binding of mitotic FAK is 24–33% of the level shown by interphase FAK, while FAK from trypsinized cells showed 65–85% binding of the interphase level (Fig. 5 b). The binding of paxillin to SP1 beads, in contrast, revealed no difference between mitotic and interphase cells (data not shown).

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