Phosphatases Generate Signal Specificity Downstream of Ssp1 Kinase in Fission Yeast

Abstract

AMPK-related protein kinases (ARKs) coordinate cell growth, proliferation, and migration with environmental status. It is unclear how specific ARKs are activated at specific times. In the fission yeast Schizosaccharomyces pombe, the CaMKKlike protein kinase Ssp1 promotes cell cycle progression by activating the ARK Cdr2 according to cell growth signals. Here, we demonstrate that Ssp1 activates a second ARK, Ssp2/AMPK␣, for cell proliferation in low environmental glucose. Ssp1 activates these two related targets by the same biochemical mechanism: direct phosphorylation of a conserved residue in the activation loop (Cdr2-T166 and Ssp2-T189). Despite a shared upstream kinase and similar phosphorylation sites, Cdr2 and Ssp2 have distinct regulatory input cues and distinct functional outputs. We investigated this specificity and found that distinct protein phosphatases counteract Ssp1 activity toward its different substrates. We identified the PP6 family phosphatase Ppe1 as the primary phosphatase for Ssp2-T189 dephosphorylation. The phosphatase inhibitor Sds23 acts upstream of PP6 to regulate Ssp2-T189 phosphorylation in a manner that depends on energy but not on the intact AMPK heterotrimer. In contrast, Cdr2-T166 phosphorylation is regulated by protein phosphatase 2A but not by the Sds23-PP6 pathway. Thus, our study provides a phosphatase-driven mechanism to induce specific physiological responses downstream of a master protein kinase.KEYWORDS AMPK, Cdr2, Ssp1, fission yeast, kinase, phosphatase, pombe C ells control proliferation, polarized growth, and migration according to nutrient and environmental status. These processes must be tightly regulated during normal growth and development, and loss of this coordination is coupled with distinct steps in the initiation and maturation of tumors (1, 2). The AMP-activated protein kinase (AMPK)-related kinase (ARK) subfamily of protein kinases is essential for coordination of cell growth, proliferation, and migration with environmental status (3). Human cells express at least 13 members of this conserved kinase subfamily, including the metabolic sensor AMPK, the cytoskeletal kinase MARK1/Par-1, and the SAD kinases that regulate cell growth and polarity (4). Given their functions in diverse cell biological processes, it is remarkable that all ARKs can be activated by a shared upstream activating kinase, which phosphorylates the activation loop of ARK kinase domains to turn them "on" (4). To meet the needs of a rapidly changing environment, different ARKs need to be turned on and off at different times. This leads to a simple question: how does a common activation mechanism activate different ARKs at different times?The defining member of the ARK subfamily is AMPK. This conserved heterotrimeric complex is activated by low cellular energy status, typically the result of low levels of environmental nutrients (5). AMPK activation shifts cells toward catabolism and promotes the Warburg effect in cancer cells (5-7). The AMPK heterotrimer consists of a catalytic ␣ subun...

The phosphatase inhibitor Sds23 regulates cell division symmetry in fission yeast

“…Both Sds23 and Igo1 are known to be hyperphosphorylated, which may serve to regulate their interactions with PP2A (Hanyu et al, 2009;Chica et al, 2016). We also note that the Ppk18-Igo1 pathway is upregulated during nitrogen stress (Chica et al, 2016), and Sds23 functions in pathways upregulated during glucose depletion and energy stress (Yakura et al, 2006;Hanyu et al, 2009;Jang et al, 2013;Saitoh et al, 2015;Deng et al, 2017). Thus, it will be interesting to determine how these pathways contribute to cytokinesis when cells encounter environmental stresses linked to their functions.…”

“…The partially overlapping roles of Sds23 and Igo1, as shown by synthetic genetic defects, raise possibilities for dynamic control of cytokinesis by PP2A during cell cycle progression. The Greatwall-endosulfine pathway including Ppk18-Igo1 is best known for regulation of mitotic entry (Mochida et al, 2010;Burgess et al, 2010;Chica et al, 2016), and Sds23 has also been linked to cell cycle progression through phenotypes such as cell size (Ishii et al, 1996;Jang et al, 1997;Deng et al, 2017). These functional connections to the cell cycle suggest that timely regulation of PP2A by these proteins could ensure proper cytokinesis.…”

“…In this study, we have identified and characterized cytokinesis defects in cells lacking Sds23, which binds to and inhibits PP2A-related phosphatases (Hanyu et al, 2009;Deng et al, 2017). We found that the CAR assembles off-center in sds23∆ mutants, and once assembled slides away from the cell middle due to delayed recruitment of potential anchors.…”

“…While studying Sds23 in nutrient signaling (Deng et al, 2017), we observed that sds23∆ cells divide with off-center septa ( Figure 1A). We quantified this defect by measuring the Cell…”

“…Sds23 physically associates with PP2A and PP6 phosphatases, and can inhibit PP6 phosphatase activity (Hanyu et al, 2009;Deng et al, 2017). Similar to other organisms, the fission yeast endosulfine protein Igo1 inhibits PP2A activity (Chica et al, 2016).…”