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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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Reversal of mobility shifts of FAK, CAS, and paxillin  (PAX) during post-mitotic cell spreading. Mobility shifts were  examined by immunoblotting of total cell lysates prepared from  interphase (I) or mitotic (M) cells, or cells released from mitotic  arrest (numbered lanes, n = min removed from nocodazole). Total cell lysates were blotted on PVDF membranes, and the membranes were probed with the antibodies against CAS, FAK, paxillin, or cyclin B, as indicated. Cyclin B1 immunoblot is shown as  an indicator of metaphase–anaphase transition. Note that CAS,  FAK, and paxillin show reversal of mobility shifts during 80–180  min after the release of mitotic arrest, a time span corresponding  to post-mitotic cell spreading.
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Figure 6: Reversal of mobility shifts of FAK, CAS, and paxillin (PAX) during post-mitotic cell spreading. Mobility shifts were examined by immunoblotting of total cell lysates prepared from interphase (I) or mitotic (M) cells, or cells released from mitotic arrest (numbered lanes, n = min removed from nocodazole). Total cell lysates were blotted on PVDF membranes, and the membranes were probed with the antibodies against CAS, FAK, paxillin, or cyclin B, as indicated. Cyclin B1 immunoblot is shown as an indicator of metaphase–anaphase transition. Note that CAS, FAK, and paxillin show reversal of mobility shifts during 80–180 min after the release of mitotic arrest, a time span corresponding to post-mitotic cell spreading.

Mentions: We examined at which stages of cell division mitosis-specific phosphorylation reversed, because such information would be useful to conceive the physiological roles of mitosis-specific phosphorylation. Nocodazole-arrested mitotic cells were collected and released to allow entry into G1, and total cell lysates were prepared at different stages of metaphase-G1 transition. The mobilities of FAK, CAS, and paxillin were analyzed by immunoblotting. As Fig. 6 shows, the mobility shifts of FAK, CAS, and paxillin continue for 80–120 min after the release of metaphase arrest, with FAK exhibiting the fastest recovery. At 120 min FAK mobility appeared normal, while significant amounts of paxillin and CAS still retained slower mobilities. It should be noted that this time point (120 min) corresponded to post-mitotic cell spreading, long after cytokinesis (which occurred during 40–60 min). Metaphase–anaphase transition was confirmed by a cyclin B immunoblot. The cyclin B band started to disappear at 40 min (Fig. 6).


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)

Reversal of mobility shifts of FAK, CAS, and paxillin  (PAX) during post-mitotic cell spreading. Mobility shifts were  examined by immunoblotting of total cell lysates prepared from  interphase (I) or mitotic (M) cells, or cells released from mitotic  arrest (numbered lanes, n = min removed from nocodazole). Total cell lysates were blotted on PVDF membranes, and the membranes were probed with the antibodies against CAS, FAK, paxillin, or cyclin B, as indicated. Cyclin B1 immunoblot is shown as  an indicator of metaphase–anaphase transition. Note that CAS,  FAK, and paxillin show reversal of mobility shifts during 80–180  min after the release of mitotic arrest, a time span corresponding  to post-mitotic cell spreading.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132894&req=5

Figure 6: Reversal of mobility shifts of FAK, CAS, and paxillin (PAX) during post-mitotic cell spreading. Mobility shifts were examined by immunoblotting of total cell lysates prepared from interphase (I) or mitotic (M) cells, or cells released from mitotic arrest (numbered lanes, n = min removed from nocodazole). Total cell lysates were blotted on PVDF membranes, and the membranes were probed with the antibodies against CAS, FAK, paxillin, or cyclin B, as indicated. Cyclin B1 immunoblot is shown as an indicator of metaphase–anaphase transition. Note that CAS, FAK, and paxillin show reversal of mobility shifts during 80–180 min after the release of mitotic arrest, a time span corresponding to post-mitotic cell spreading.
Mentions: We examined at which stages of cell division mitosis-specific phosphorylation reversed, because such information would be useful to conceive the physiological roles of mitosis-specific phosphorylation. Nocodazole-arrested mitotic cells were collected and released to allow entry into G1, and total cell lysates were prepared at different stages of metaphase-G1 transition. The mobilities of FAK, CAS, and paxillin were analyzed by immunoblotting. As Fig. 6 shows, the mobility shifts of FAK, CAS, and paxillin continue for 80–120 min after the release of metaphase arrest, with FAK exhibiting the fastest recovery. At 120 min FAK mobility appeared normal, while significant amounts of paxillin and CAS still retained slower mobilities. It should be noted that this time point (120 min) corresponded to post-mitotic cell spreading, long after cytokinesis (which occurred during 40–60 min). Metaphase–anaphase transition was confirmed by a cyclin B immunoblot. The cyclin B band started to disappear at 40 min (Fig. 6).

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