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  current news   Press   selected story    
     
  17th Aug 2012  
  Cdc28-Cln3 phosphorylation of Sla1 regulates actin patch dynamics in different modes of fungal growth
 
 




Authors
Guisheng Zeng, Yan-Ming Wang, and Yue Wang.

Published in Molecular Biology of the Cell on 11 July 2012

Abstract
A dynamic balance between targeted transport and endocytosis is critical for polarized cell growth. However, how actin-mediated endocytosis is regulated in different growth modes remains unclear. Here, we report differential regulation of cortical actin patch dynamics between the yeast and hyphal growth in Candida albicans. The mechanism involves phospho-regulation of the endocytic protein Sla1 by the cyclin-dependent kinase (CDK) Cdc28−Cln3 and the actin-regulating kinase Prk1. Mutational studies of the CDK phosphorylation sites of Sla1 revealed that Cdc28−Cln3 phosphorylation of Sla1 enhances its further phosphorylation by Prk1, weakening Sla1 association with Pan1, an activator of the actin-nucleating Arp2/3 complex. Sla1 is rapidly dephosphorylated upon hyphal induction and remains so throughout hyphal growth. Consistently, cells expressing a phosphomimetic version of Sla1 exhibited markedly reduced actin patch dynamics, impaired endocytosis, and defective hyphal development, while a nonphosphorylatable Sla1 had the opposite effect. Together, our findings establish a molecular link between CDK and a key component of the endocytic machinery, revealing a novel mechanism by which endocytosis contributes to cell morphogenesis.


Figure Legend:
Phosphorylation of Sla1 by Cdc28−Cln3. (A) Schematic diagram of Sla1 showing CDK phosphorylation sites (vertical lines). Solid boxes indicate SH3 domains and hatched boxes show putative Prk1 kinase recognition motifs. (B) Detection of phosphorylated Sla1 in vivo. Sla1-HA was immunoprecipitated from GZY584 (SLA1-HA) yeast cell lysate and subjected to λPP or mock treatment, followed by αHA WB analysis. (C) Purification of Sla1 for phospho-site mapping. HA-Sla1 was immunoprecipitated from YPG culture of GZY631 (PGAL1-HA-SLA1) yeast cells and electrophoresed on SDS-PAGE. After staining with CBB, the Sla1 band was excised for phospho-site mapping by MS. (D) Cln3-dependent phosphorylation of Sla1 in vivo. GMM culture of cln3-sd yeast cells expressing Sla1-HA (GZY622) was incubated with 0.5 mM Met and Cys (MC) at 30°C for 2 h to switch off CLN3 expression. Sla1-HA was immunoprecipitated from cell lysates for WB analysis. A WT strain (BWP17) expressing Sla1-HA (GZY584) was included as a control, and half of the immunoprecipitated Sla1-HA was treated with λPP. (E) Sla1 phosphorylation in Cln3-depleted cells. GZY622 cells were grown in GMM in the presence of 0.5 mM MC at 30°C and samples were taken at 15-min intervals for IP and WB analysis of Sla1-HA using αHA. Part of Sla1-HA from the 0-min time point was treated with λPP for control. (F) Comparison of Sla1 phosphorylation between ccn1Δ/Δ and WT cells. Sla1-HA was immunoprecipitated from lysates of WT (GZY584) and ccn1Δ/Δ (GZY744) yeast cells, and subjected to λPP or mock treatment for WB analysis. (G) Comparison of Sla1 phosphorylation between hgc1Δ/Δ and WT cells. WT (GZY584) and hgc1Δ/Δ (GZY624) yeast cells were induced with 20% FBS at 37°C for 2 h for hyphal growth. Sla1-HA was then immunoprecipitated and subjected to λPP or mock treatment for WB analysis. (H) In vitro phosphorylation of Sla1 by Cdc28−Cln3. Cln3-Myc was immunoprecipitated from GZY641 (cdc28as CLN3-Myc) yeast cells and incubated with immuno-purified HA-Sla1 to perform in vitro kinase assay in the presence or absence of 25 μM 1NMPP1. Immunoprecipitates from IS89 (cdc28as) cell lysates were used as a negative control. After electrophoresis, the gel was stained with CBB to visualize HA-Sla1 and dried onto a filter paper for autoradiography.



For more information on Yue WANG's laboratory, please here.