Fission yeast Pak1 phosphorylates anillin-like Mid1 for spatial control of cytokinesis

Abstract

Protein kinases direct polarized growth by regulating the cytoskeleton in time and space and could play similar roles in cell division. We found that the Cdc42-activated polarity kinase Pak1 colocalizes with the assembling contractile actomyosin ring (CAR) and remains at the division site during septation. Mutations in pak1 led to defects in CAR assembly and genetic interactions with cytokinesis mutants. Through a phosphoproteomic screen, we identified novel Pak1 substrates that function in polarized growth and cytokinesis. For cytokinesis, we found that Pak1 regulates the localization of its substrates Mid1 and Cdc15 to the CAR. Mechanistically, Pak1 phosphorylates the Mid1 N-terminus to promote its association with cortical nodes that act as CAR precursors. Defects in Pak1-Mid1 signaling lead to misplaced and defective division planes, but these phenotypes can be rescued by synthetic tethering of Mid1 to cortical nodes. Our work defines a new signaling mechanism driven by a cell polarity kinase that promotes CAR assembly in the correct time and place.

Pak1 kinase controls cell shape through ribonucleoprotein granules

“…This phenotype represents a partial orb shape, confirming its role in this process but also indicating that additional factors contribute to the complete orb shape defect. Consistent with this possibility, both Pak1 and Orb6 have additional substrates within the Cdc42 and exocytosis networks (Chang et al, 1999; Das et al, 2015; Magliozzi et al, 2020; Tay et al, 2019). Pak1 and Orb6 may control cell polarity through coordinated regulation of RNPs and Cdc42 signaling, or alternatively by acting independently on these two downstream targets.…”

“…We recently performed an unbiased phosphoproteomic screen that identified Sts5 as a leading candidate for phosphorylation by Pak1 in fission yeast cells (Magliozzi et al, 2020). Specifically, phosphorylation of Sts5 residues S261 and S264 was reduced 2.5-fold in pak1-as , a partial loss-of-function mutant, compared to wild type cells (Loo and Balasubramanian, 2008; Magliozzi et al, 2020). This phosphorylation was further reduced 2.3-fold by 15-minute addition of 3-Brb-PP1, which completely inhibits pak1-as kinase activity (Magliozzi et al, 2020).…”

“…While Cdc42 may transiently activate Pak1 at granules, several lines of evidence indicate that Pak1 could be active at granules without Cdc42. First, we and other groups have shown that recombinant full-length Pak1 expressed and purified from bacteria displays strong kinase activity in vitro (Das et al, 2012; Loo and Balasubramanian, 2008; Magliozzi et al, 2020; Pollard et al, 2017; Yang et al, 1999), which means that it is not autoinhibited under these conditions. Second, PAKs can also be activated by various classes of lipids and proteins (Bokoch et al, 1998; Strochlic et al, 2010; Zenke et al, 1999).…”

“…Active Pak1 regulates components of the Cdc42 network and the cytoskeleton (Edwards et al, 1999; Moshfegh et al, 2014; Vadlamudi et al, 2005, 2002). Beyond these canonical targets, a recent phosphoproteomic screen identified potential substrates that suggest novel mechanisms for Pak1 function in cell morphology and viability (Magliozzi et al, 2020).…”

The Orb6-Sts5 Axis Regulates Stress Granule Formation and Heat Stress Response in Fission Yeast

“…For example, other factors such as the vigilin homologue Vgl1 (Wen et al ., 2010), signaling factors such as calcineurin (Higa et al ., 2015), and protein kinase C Pck2 (Kanda et al ., 2021) also associate with stress granules in response to thermal stress, and stress granule formation is also stimulated in response to heat stress by the RNA-binding protein Nrd1 (Satoh et al ., 2012). Additionally, the intrinsically disordered N-terminus of Sts5 is phosphorylated on multiple residues, suggesting it is the target of additional protein kinases (Magliozzi et al ., 2020).…”