Phosphorylation by CDK1 regulates XMAP215 function in vitro.
XMAP215, a microtubule-associated protein isolated from Xenopus eggs, promotes microtubule assembly dynamics in an end-specific manner: addition of XMAP215 to purified porcine tubulin increases both elongation and shortening rates at microtubule plus ends, with minimal effects at minus ends. Previous results indicated that XMAP215 is phosphorylated during M phase, suggesting that its activity may be regulated by cell cycle phosphorylation. To test this hypothesis, we used video-enhanced DIC microscopy to examine the effects of XMAP215 phosphorylated by CDK1 on the assembly of purified tubulin. XMAP215 incubated with ATP at 30 degrees C for 10- 20 min in the absence of CDK1 exhibited a 4.1-fold increase in plus end elongation rate compared to purified tubulin. Elongation was promoted to a lesser degree (2.4-fold) by phosphorylated XMAP215. In contrast, XMAP215 phosphorylation did not alter the approximately 3-fold increase in shortening rate. XMAP215 binding to taxol microtubules was also not changed by phosphorylation. To further investigate mechanisms responsible for the faster microtubule shortening rate observed with XMAP215, we made microtubules with segments assembled prior to XMAP215 addition (proximal segments) and segments assembled in the presence of XMAP215 (distal segments). In 9 of 10 microtubules, the distal segment shortened faster (distal = 60.7 microm/min; proximal = 37.5 microm/min), suggesting that MTs assembled in the presence of XMAP215 have an altered lattice that results in subsequent faster shortening.
Vasquez
R J
RJ
Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015, USA.
Gard
D L
DL
Cassimeris
L
L
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002457:Cell Extracts; D008869:Microtubule-Associated Proteins / Q000378:metabolism* / Q000502:physiology; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D010063:Ovum; D010766:Phosphorylation; D029867:Xenopus Proteins; D014982:Xenopus laevis
C486738:CKAP5 protein, Xenopus; D002457:Cell Extracts; D008869:Microtubule-Associated Proteins; D029867:Xenopus Proteins; D016203:CDC2 Protein Kinase