Cell cycle-dependent phosphorylation of the 77 kDa echinoderm microtubule-associated protein (EMAP) in vivo and association with the p34cdc2 kinase

Abstract

The most abundant microtubule-associated protein in sea urchin eggs and embryos is the 77 kDa echinoderm microtubule-associated protein (EMAP). EMAP localizes to the mitotic spindle as well as the interphase microtubule array and is a likely target for a cell cycle-activated kinase. To determine if EMAP is phosphorylated in vivo, sea urchin eggs and embryos were metabolically labeled with 32PO4 and a monospecific antiserum was used to immunoprecipitate EMAP from 32P-labeled eggs and embryos. In this study, we demonstrate that the 77 kDa EMAP is phosphorylated in vivo by two distinct mechanisms. In the unfertilized egg, EMAP is constitutively phosphorylated on at least five serine residues. During the first cleavage division following fertilization, EMAP is phosphorylated with a cell cycle-dependent time course. As the embryo enters mitosis, EMAP phosphorylation increases, and as the embryo exits mitosis, phosphorylation decreases. During mitosis, EMAP is phosphorylated on 10 serine residues and two-dimensional phosphopeptide mapping reveals a mitosis-specific site of phosphorylation. At all stages of the cell cycle, a 33 kDa polypeptide copurifies with the 77 kDa EMAP, regardless of phosphorylation state. Antibodies against the cdc2 kinase were used to demonstrate that the 33 kDa polypeptide is the p34cdc2 kinase. The p34cdc2 kinase copurifies with the mitotic apparatus and immunostaining indicates that the p34cdc2 kinase is concentrated at the spindle poles. Models for the interaction of the p34cdc2 kinase and the 77 kDa EMAP are presented.

Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression

“…However, we do not exclude the possibility that EML4 may also be subject to additional phospho-dependent regulation by other mitotic, as well as nonmitotic, kinases. The sea urchin EMAP protein was found to be efficiently phosphorylated by CDK1 (28). We also observed loss of the gel mobility shift of the EML4-NTD in mitosis upon addition of a CDK1 inhibitor, but this result can be explained by CDK1 acting upstream of NEK9.…”

“…This switch is largely driven through a change in the complement of MAPs associated with microtubules and major changes in microtubule nucleation capacity (26). Observations that both sea urchin EMAP and human EML4 undergo phosphorylation during mitotic progression suggest that regulation of EML proteins may contribute to these changes in the microtubule network (27,28). EML2, EML3, and EML4 were identified in a large-scale proteomic analysis of human NIMA-related kinase 6 (NEK6) binding proteins (29).…”

DCX-EMAP is a core organizer for the ultrastructure of Drosophila mechanosensory organelles

“…DCX-EMAP belongs to the EMAP (echinoderm-microtubule-associated proteins) family, the members of which are regulators for microtubule dynamics ( Brisch et al, 1996 ; Eichenmuller et al, 2002 ; Hamill et al, 1998 ; Pollmann et al, 2006 ). All EMAP proteins share a conserved TAPE (tandem atypical propeller in EMLs) domain at the carboxyl-terminal, including the HELP (hydrophobic echinoderm-microtubule-associated-like protein) domain and multiple WD40 repeats.…”