Phosphorylation by p34cdc2 protein kinase regulates binding of the kinesin-related motor HsEg5 to the dynactin subunit p150.
The kinesin-related motor HsEg5 is essential for centrosome separation, and its association with centrosomes appears to be regulated by phosphorylation of tail residue threonine 927 by the p34(cdc2) protein kinase. To identify proteins able to interact with the tail of HsEg5, we performed a yeast two-hybrid screen with a HsEg5 stalk-tail construct as bait. We isolated a cDNA coding for the central, alpha-helical region of human p150(Glued), a prominent component of the dynactin complex. The interaction between HsEg5 and p150(Glued) was enhanced upon activation of p34(CDC28), the budding yeast homolog of p34(cdc2), provided that HsEg5 had a phosphorylatable residue at position 927. Phosphorylation also enhanced the specific binding of p150(Glued) to the tail domain of HsEg5 in vitro, indicating that the two proteins are able to interact directly. Immunofluorescence microscopy revealed co-localization of HsEg5 and p150(Glued) during mitosis but not during interphase, consistent with a cell cycle-dependent association between the two proteins. Taken together, these results suggest that HsEg5 and p150(Glued) may interact in mammalian cells in vivo and that p34(cdc2) may regulate this interaction. Furthermore, they imply that the dynactin complex may functionally interact not only with dynein but also with kinesin-related motors.
Blangy
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Swiss Institute for Experimental Cancer Research (ISREC), 155, Chemin des Boveresses, CH-1066 Epalinges, Switzerland.
Arnaud
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Nigg
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EA
D000595:Amino Acid Sequence; D000818:Animals; D016203:CDC2 Protein Kinase / Q000502:physiology*; D000072159:Dynactin Complex; D006367:HeLa Cells; D006801:Humans; D016547:Kinesins / Q000502:physiology*; D008869:Microtubule-Associated Proteins / Q000502:physiology*; D008938:Mitosis; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011817:Rabbits
C000606360:DCTN1 protein, human; D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D016203:CDC2 Protein Kinase; D016547:Kinesins