Mitotic regulation of protein 4.1R involves phosphorylation by cdc2 kinase.
The nonerythrocyte isoform of the cytoskeletal protein 4.1R (4.1R) is associated with morphologically dynamic structures during cell division and has been implicated in mitotic spindle function. In this study, we define important 4.1R isoforms expressed in interphase and mitotic cells by RT-PCR and mini-cDNA library construction. Moreover, we show that 4.1R is phosphorylated by p34cdc2 kinase on residues Thr60 and Ser679 in a mitosis-specific manner. Phosphorylated 4.1R135 isoform(s) associate with tubulin and Nuclear Mitotic Apparatus protein (NuMA) in intact HeLa cells in vivo as well as with the microtubule-associated proteins in mitotic asters assembled in vitro. Recombinant 4.1R135 is readily phosphorylated in mitotic extracts and reconstitutes mitotic aster assemblies in 4.1R-immunodepleted extracts in vitro. Furthermore, phosphorylation of these residues appears to be essential for the targeting of 4.1R to the spindle poles and for mitotic microtubule aster assembly in vitro. Phosphorylation of 4.1R also enhances its association with NuMA and tubulin. Finally, we used siRNA inhibition to deplete 4.1R from HeLa cells and provide the first direct genetic evidence that 4.1R is required to efficiently focus mitotic spindle poles. Thus, we suggest that 4.1R is a member of the suite of direct cdc2 substrates that are required for the establishment of a bipolar spindle.
Huang
Shu-Ching
SC
Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA 02115, USA. shu-ching_huang@dfci.harvard.edu
Liu
Eva S
ES
Chan
Siu-Hong
SH
Munagala
Indira D
ID
Cho
Heidi T
HT
Jagadeeswaran
Ramasamy
R
Benz
Edward J
EJ
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism / Q000502:physiology*; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D008938:Mitosis; D008969:Molecular Sequence Data; D010641:Phenotype; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D008941:Spindle Apparatus / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D014404:Tubulin; C028841:erythrocyte membrane band 4.1 protein