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...

Transient activation of the cellular energy sensor AMPK during osmotic stress requires its energy-sensing subunit. Cellular ATP levels decrease during osmotic stress, which triggers energy stress, which in turn requires dynamic activation of AMPK.

The identification and quantification of important biomarkers is a critical first step in the elucidation of biological systems. Biomarkers take many forms as cellular responses to stimuli and can be manifested during transcription, translation, and/or metabolic processing. Increasingly, researchers have relied upon mixed-isotope labeling (MIL) coupled with MS to perform relative quantification of biomarkers between two or more biological samples. MIL effectively tags biomarkers of interest for ease of identification and quantification within the mass spectrometer by using isotopic labels that introduce a heavy and light form of the tag. In addition to MIL coupled with MS, a number of other approaches have been used to quantify biomarkers including protein gel staining, enzymatic labeling, metabolic labeling, and several label-free approaches that generate quantitative data from the MS signal response. This review focuses on MIL techniques coupled with MS for the quantification of protein and small-molecule biomarkers.

Kinesin-binding protein inhibits kinesin activity through structural alterations of the kinesin motor domain.

Animal and fungal cells divide through the assembly, anchoring, and constriction of a contractile actomyosin ring (CAR) during cytokinesis. The timing and position of the CAR must be tightly controlled to prevent defects in cell division, but many of the underlying signaling events remain unknown. The conserved heterotrimeric protein phosphatase PP2A controls the timing of events in mitosis, and upstream pathways including Greatwall–Ensa regulate PP2A activity. A role for PP2A in CAR regulation has been less clear, although loss of PP2A in yeast causes defects in cytokinesis. Here, we report that Sds23, an inhibitor of PP2A family protein phosphatases, promotes the symmetric division of fission yeast cells through spatial control of cytokinesis. We found that sds23∆ cells divide asymmetrically due to misplaced CAR assembly, followed by sliding of the CAR away from its assembly site. These mutant cells exhibit delayed recruitment of putative CAR anchoring proteins including the glucan synthase Bgs1. Our observations likely reflect a broader role for regulation of PP2A in cell polarity and cytokinesis because sds23∆ phenotypes were exacerbated when combined with mutations in the fission yeast Ensa homologue, Igo1. These results identify the PP2A regulatory network as a critical component in the signaling pathways coordinating cytokinesis.