Phosphorylation by p34cdc2 Protein Kinase Regulates Binding of the Kinesin-related Motor HsEg5 to the Dynactin Subunit p150Glued

Anne Blangy,Lionel Arnaud,Erich A Nigg

doi:10.1074/jbc.272.31.19418

Abstract

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.

Highlights

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 p34cdc2 protein kinase
We showed that HsEg5 associates with centrosomes in early prophase [43], and data obtained for both XlEg5 and HsEg5 indicate that this event depends on phosphorylation of tail residue threonine 927 (Thr-927) by p34cdc2/cyclin B [43, 44]
The distributions of HsEg5 and p150Glued overlap at least partially as soon as mitosis begins (Fig. 4, c– e). Both proteins remain associated with the mitotic spindle throughout mitosis (Fig. 4, f– k). These immunocytochemical data support the idea that HsEg5 and p150Glued may interact in yeast cells and in vitro and in mammalian cells in vivo; they are consistent with the hypothesis that the interaction between the two proteins is controlled by cell cycle-dependent phosphorylation of Thr-927 in the tail domain of HsEg5

Summary

Introduction

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 p34cdc2 protein kinase. Phosphorylation enhanced the specific binding of p150Glued to the tail domain of HsEg5 in vitro, indicating that the two proteins are able to interact directly.

Results

Conclusion