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.
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.
Ookata
K
K
Laboratory of Cell and Developmental Biology, Faculty of Biosciences, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226, Japan.
Hisanaga
S
S
Sugita
M
M
Okuyama
A
A
Murofushi
H
H
Kitazawa
H
H
Chari
S
S
Bulinski
J C
JC
Kishimoto
T
T
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000494:pharmacology; D001665:Binding Sites; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002453:Cell Cycle; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis / Q000502:physiology; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism
D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; D010768:Phosphoserine; C082868:butyrolactone I; D016203:CDC2 Protein Kinase; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D015107:4-Butyrolactone; D013936:Thymidine