Serum-dependent phosphorylation of human MAP4 at Ser696 in cultured mammalian cells

In the previous paper {Ookata et al., (1997) Biochemistry, 36: 249-259}, we identified two mitotic cdc2 kinase phosphorylation sites (Ser696 and Ser78 7) in the proline-rich region of human MAP4. One (Ser696) of them was also phosphorylated during interphase. A protein kinase responsible for interpha se phosphorylation of Ser696 could necessarily be distinct from cdc2/cyclin B kinase, To get insights into a physiological role for Ser696 phosphoryla tion, we searched for a Ser696 kinase and for cellular conditions under whi ch Ser696 is dephosphorylated. Because Ser696 conforms to the MAP kinase ph osphorylation consensus motif (PXSP), MAP kinase was tested as a possible k inase phosphorylating Ser696. MAP kinase, in fact, did phosphorylate Ser696 in MTB3, the carboxy-terminal half of human MAP4 in vitro. Phosphorylation of Ser696 in HeLa cell extract was suppressed by a MAP kinase inhibitor, D BTM-0004. Also consistent with the notion that Ser696 is a MAP kinase site were the fact that serum-starvation induced dephosphorylation of Ser696 in HeLa cells, TIG-3 and MRC-5-30 human fibroblasts, while readdition of serum recovered Ser696 phosphorylation, albeit after a surprisingly long interva l. Thus, phosphorylation of Ser696 of MAP4, most likely carried out by MAP kinase, may play a role in modulation of MAP4 activity in proliferating ver sus quiescent cells.