MAP4 Is the in Vivo Substrate for CDC2 Kinase in HeLa Cells:  Identification of an M-Phase Specific and a Cell Cycle-Independent Phosphorylation Site in MAP4

Abstract

We reported previously that cdc2 kinase decreased the microtubule-stabilizing ability of a major HeLa cell microtubule-associated protein, MAP4, by phosphorylation in vitro [Ookata, K., et al. (1995) J. Cell Biol. 128, 849-862]. An important question raised by this study is whether MAP4 is indeed phosphorylated by cdc2 kinase at mitosis in vivo. We present here evidence that cdc2 kinase is the major M-phase MAP4 kinase, and, further, we identify two phosphorylation sites within the proline-rich domain of MAP4. Metabolic 32P labeling showed the increased phosphorylation of MAP4 at mitosis. A specific inhibitor of cdc2 kinase, butyrolactone I, inhibited phosphorylation of MAP4 both in mitotic HeLa cells and in the mitotic HeLa cell extract. The phosphopeptide map analysis revealed the high similarity of in vivo labeled mitotic MAP4 to that phosphorylated by cdc2 kinase in vitro. Ser-696 and Ser-787, both of which lie within SPXK consensus sequences for cdc2 kinase, were identified as phosphorylation sites in the proline-rich region of MAP4 in vivo and in vitro. Immunoblotting with antibodies that recognize the phosphorylation state of Ser-696 or Ser-787 showed that Ser-787 in the SPSK sequence was specifically phosphorylated at mitosis while Ser-696 in the SPEK sequence was phosphorylated both at mitosis and in interphase. These results suggest that cdc2 kinase directly regulates microtubule dynamics at mitosis through phosphorylation of MAP4 at a number of sites, including Ser-787.

Mammalian Septins Regulate Microtubule Stability through Interaction with the Microtubule-binding Protein MAP4

“…Cdc2 kinase phosphorylates at least two serines in the PRD (Ookata et al, 1997;Kitazawa et al, 2000), whereas kinases of the MARK/Par-1 family phosphorylate multiple serines in the AD (Drewes et al, 1997(Drewes et al, , 1998Ebneth et al, 1999). These phosphorylation events inhibit the MAP4 -MT interaction and lead to destabilization of MTs (Drewes et al, 1997;Drewes et al, 1998;Ebneth et al, 1999).…”

“…The proline-rich domain (PRD, aa 654 -895) contains at least one MT binding site, as well as a region that promotes MT assembly and bundling (Aizawa et al, 1991). This region also binds to cyclin B1 and contains at least two cdk2 phosphorylation sites (Ookata et al, 1995(Ookata et al, , 1997Kitazawa et al, 2000). The C-terminal affinity domain (AD, aa 902-1090) contains up to four motifs (depending on the isoform) that are phosphorylated by MARK/Par-1 family kinases.…”

Mitotic kinase Aurora-A phosphorylates RASSF1A and modulates RASSF1A-mediated microtubule interaction and M-phase cell cycle regulation

“…Interestingly, p34/cdc2-induced APC phosphorylation has been found to disrupt APC and microtubule interactions, a finding that highlights the importance of phosphorylation in regulation of APC interactions with microtubules (Trzepacz et al, 1997). MAP4 is another important microtubule-binding protein that has been found to be phosphorylated by mitotic kinases including p34/cdc2 and microtubule-affinity regulating kinases (Illenberger et al, 1996;Ookata et al, 1997). Evidence suggests that MAP4 phosphorylation induced by these mitotic kinases causes MAP4 to dissociate from microtubules and thereby increase microtubule dynamics during mitosis (Illenberger et al, 1996).…”

“…Previous studies have shown that microtubule-binding/associated proteins (MAPs) such as MAP2 and MAP4 are phosphorylated during mitosis (Faruki et al, 1992;Ookata et al, 1997). Phosphorylation is a critical event regulating these MAPs as it causes their dissociation from microtubules and thereby facilitates M-phase cell cycle progression.…”

Ser787 in the Proline-Rich Region of Human MAP4 is a Critical Phosphorylation Site that Reduces its Activity to Promote Tubulin Polymerization.

“…This dynamic property of mitotic MTs is thought to be caused, at least in part, by the decreased MT-stabilizing ability of MAP4 at mitosis, which could be induced by phosphorylation with p34 cdc2 /cyclin B kinase. MAP4 was shown to be phosphorylated by p34 cdc2 kinase in HeLa cells (Ookata et al, 1997), and its phosphorylation was shown to reduce MT polymerizing ability in vitro Ookata et al, 1995). In Xenopus eggs, too, mitotic phosphorylation of a MAP4-like protein (XMAP230 or p220) was shown to participate in mitotic control of MT dynamics (Shiina et al, 1992;Andersen et al, 1994;Faruki and Karsenti, 1994).…”