Phosphorylation of MAP4 affects microtubule properties and cell cycle progression.
In human cells, MAP4, a microtubule-associated protein ubiquitously expressed in proliferating cells, has been shown to undergo in vivo phosphorylation. Two phosphorylation sites, serines 696 and 787, lie within the proline-rich region of its microtubule-binding domain. To test the hypothesis that phosphorylation at these sites influences microtubule properties or cell cycle progression, we prepared stable cell lines that inducibly express versions of MAP4 in which phosphorylation of these two serines was prevented by their replacement with alanine, lysine, or glutamate residues (AA-, KK-, or EE-MAP4). All nonphosphorylatable mutant forms of MAP4 expressed in mouse Ltk- cells were localized to MT arrays that were unremarkable in appearance. Expression of nonphosphorylatable mutants of MAP4 did not affect cell doubling time; however, expression of some mutants altered progression into or through cell division. Interactions of mutant MAP4 with MTs were examined in vitro. KK mutant MAP4 bound MTs more avidly than its wild-type counterpart, WT-MAP4. In vivo MT polymer also differed among the mutants: MTs in cells expressing the KK- and AA-MAP4 forms were more resistant to nocodazole depolymerization than those in cells expressing EE- or WT-MAP4 forms. Our results demonstrate that phosphorylation alters MAP4 properties and suggest a raison d'être for phosphorylation of the MAP4 microtubule-binding domain during cell cycle progression.
Chang
W
W
Department of Biological Sciences, College of Arts & Sciences, Columbia University, New York, NY 10027-2450, USA.
Gruber
D
D
Chari
S
S
Kitazawa
H
H
Hamazumi
Y
Y
Hisanaga
S
S
Bulinski
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001665:Binding Sites / Q000502:physiology; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D019926:Cyclin B / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D015870:Gene Expression / Q000502:physiology; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008938:Mitosis / Q000187:drug effects / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism
D000970:Antineoplastic Agents; D016895:Culture Media, Serum-Free; D019926:Cyclin B; D008869:Microtubule-Associated Proteins; D012694:Serine; D016203:CDC2 Protein Kinase; D015739:Nocodazole