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Septin-associated protein kinase Hsl1 phosphorylates Pah1 to inhibit phosphatidate phosphatase activity and regulate lipid synthesis in Saccharomyces cerevisiae

ABSTRACT: In Saccharomyces cerevisiae, Pah1 phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, plays a key role in utilizing PA for the synthesis of membrane phospholipids or the neutral lipid triacylglycerol. Low activity favors phospholipid synthesis, whereas high activity favors triacylglycerol synthesis. Pah1 function is controlled by its subcellular location as regulated by phosphorylation and dephosphorylation. Multiple protein kinases phosphorylate Pah1 for its inactivation in the cytosol; Pah1 is activated via recruitment and dephosphorylation by the protein phosphatase Nem1-Spo7 at the nuclear/endoplasmic reticulum membrane where the PA phosphatase reaction occurs. Additionally, phosphorylation inhibits PA phosphatase activity and stabilizes Pah1 abundance, while dephosphorylation stimulates activity and destabilizes the enzyme abundance. Many of the protein kinases that phosphorylate Pah1 have yet to be characterized and their sites of phosphorylation defined. Here, we established Pah1 as a bona fide substrate of septin-associated Hsl1, a protein kinase involved in mitotic morphogenesis checkpoint signaling. Using Pah1 as substrate, Hsl1 activity was dependent on reaction time and the amounts of protein kinase, Pah1, and ATP. The phosphorylation occurred on Ser-748 and Ser-773, which together caused a 5-fold reduction in PA phosphatase catalytic efficiency. Analysis of cells expressing phosphorylation-deficient S748A and S773A mutant forms of Pah1 indicated that the Hsl1-mediated phosphorylation of Pah1 promoted membrane phospholipid synthesis at the expense of triacylglycerol, and ensured the dependence of Pah1 function on the Nem1-Spo7 protein phosphatase. This work advances understanding of how Hsl1 facilitates membrane phospholipid synthesis through the phosphorylation-mediated regulation of Pah1.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Saccharomyces Cerevisiae (ncbitaxon:4932)

SUBMITTER: George M. Carman

PROVIDER: MSV000095027 | MassIVE | Thu Jun 13 14:12:00 BST 2024

REPOSITORIES: MassIVE

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Project description:The model describes the double phosphorylation of MAP kinase by an ordered mechanism using the Michaelis-Menten formalism. Two different enzymes, MAPKK1 and MAPKK2, successively phosphorylate the MAP kinase, but one and the same phosphatase dephosphorylates both sites. The model reproduces figure S9 in the supplemental material of the article. The model is further described in: Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades. Markevich NI, Hoek JB, Kholodenko BN. J Cell Biol. 2004 Feb 2;164(3):353-9. PMID: 14744999 ; DOI: 10.1083/jcb.200308060 Abstract: Mitogen-activated protein kinase (MAPK) cascades can operate as bistable switches residing in either of two different stable states. MAPK cascades are often embedded in positive feedback loops, which are considered to be a prerequisite for bistable behavior. Here we demonstrate that in the absence of any imposed feedback regulation, bistability and hysteresis can arise solely from a distributive kinetic mechanism of the two-site MAPK phosphorylation and dephosphorylation. Importantly, the reported kinetic properties of the kinase (MEK) and phosphatase (MKP3) of extracellular signal-regulated kinase (ERK) fulfill the essential requirements for generating a bistable switch at a single MAPK cascade level. Likewise, a cycle where multisite phosphorylations are performed by different kinases, but dephosphorylation reactions are catalyzed by the same phosphatase, can also exhibit bistability and hysteresis. Hence, bistability induced by multisite covalent modification may be a widespread mechanism of the control of protein activity. This model originates from BioModels Database: A Database of Annotated Published Models (http://www.ebi.ac.uk/biomodels/). It is copyright (c) 2005-2010 The BioModels.net Team. For more information see the terms of use . To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.

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Septin-associated protein kinase Hsl1 phosphorylates Pah1 to inhibit phosphatidate phosphatase activity and regulate lipid synthesis in Saccharomyces cerevisiae

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