Phosphorylation by CDK1 regulates XMAP215 function in vitro

XMAP215, a microtubule-associated protein isolated from Xenopus eggs, promo tes microtubule assembly dynamics in an end-specific manner: addition of XM AP215 to purified porcine tubulin increases both elongation and shortening rates at microtubule plus ends, with minimal effects at minus ends. Previou s results indicated that XMAP215 is phosphorylated during hi phase, suggest ing that its activity may be regulated by cell cycle phosphorylation. To te st this hypothesis, we used video-enhanced DIC microscopy to examine the ef fects 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 o f CDK1 exhibited a 4.1-fold increase in plus end elongation rate compared t o purified tubulin. Elongation was promoted to a lesser degree (2.4-fold) b y phosphorylated XMAP215. In contrast, XMAP215 phosphorylation did not alte r the similar to 3-fold increase in shortening rate. XMAP215 binding to tax ol microtubules was also not changed by phosphorylation. To further investi gate mechanisms responsible for the faster microtubule shortening rate obse rved with XMAP215, we made microtubules with segments assembled prior to XM AP215 addition (proximal segments) and segments assembled in the presence o f XMAP215 (distal segments). In 9 of 10 microtubules, the distal segment sh ortened faster (distal = 60.7 mu m/min; proximal = 37.5 mu m/min), suggesti ng that MTs assembled in the presence of XMAP215 have an altered lattice th at results in subsequent faster shortening Cell Motil. Cytoskeleton 43:310- 321, 1999. (C) 1999 Wiley-Liss, Inc.