CR Li, YM Wang, Y Wang
Cyclin-dependent kinases (CDKs) drive and coordinate multiple cell-cycle events, including construction and contraction of the actomyosin ring during cytokinesis. However, it remains unclear whether CDKs regulate cytokinesis by directly targeting components of the ring. In a search for proteins containing consensus CDK phosphorylation sites in Candida albicans, we found that the IQGAP Iqg1 contains two dense clusters of 19 such sites flanking the actin-interacting CH domain. Here, we show that Iqg1 is indeed a phosphoprotein that undergoes cell-cycle-dependent phosphorylation and can be phosphorylated by purified Clb–Cdc28 kinases in vitro. Mass spectrometry identified several phosphoserine and phosphothreonine residues among these CDK sites. Mutating 15 of the CDK phosphorylation sites with alanine markedly reduced Iqg1 phosphorylation in vivo. The 15A mutation greatly stabilized Iqg1, caused both premature assembly and delayed disassembly of the actomyosin ring, blocked Iqg1 interaction with the actin-nucleating proteins Bni1 and Bnr1, and resulted in defects in cytokinesis. Our data therefore strongly support the idea that the Cdc28 CDK regulates cytokinesis partly by directly phosphorylating the actomyosin ring component Iqg1.
Figure Legend: The 15A mutation stabilizes Iqg1 and affects the dynamics of the actomyosin ring. (A) Log-phase cells expressing WT Iqg1 (LCR26), Iqg1-15A (LCR29) or Iqg1-15E (LCR32) as the sole source of Iqg1 were arrested with 30 µM nocodazole for 2 hr and then stained with rhodamine-phalloidine to reveal the actin ring at the neck and with 4',6-diamidino-2-phenylindole (DAPI) to visualize nuclei. (B) The nocodazole-arrested cells described in (A) were released into drug-free medium for growth at 30°C. Cells were harvested at intervals for actin and nuclear staining. Percentage of cells with a neck actin ring and with divided nuclei was counted. (C) Stationary cells expressing WT Iqg1 (LCR26), Iqg1-15A (LCR29) or Iqg1-15E (LCR32) were released into fresh YPD for growth at 30°C. Aliquots were harvested at 0, 1, 2, and 3 hr and lysates were prepared for Western blotting using Myc Ab (all Iqg1 proteins have a 6Myc tag). Duplicate blots were probed with PSTAIRE Ab to detect Cdc28 as loading controls. (D) Cell lysates prepared as described in (B) and analyzed by Western blotting using Myc Ab and PSTAIRE Ab.
The EMBO Journal advance online publication 16 October 2008;
For more information on A/Prof. Wang Yue’s lab, Please Click here.