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The yeast Nem1‐Spo7 phosphatase complex, which dephosphorylates and regulates Pah1 phosphatidate phosphatase, is phosphorylated by protein kinase C
Author(s) -
Dey Prabuddha,
Mirheydari Mona M,
Han GilSoo,
Carman George M
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.488.2
Subject(s) - phosphatidate , biochemistry , diacylglycerol kinase , dephosphorylation , phosphorylation , phosphatase , protein phosphorylation , kinase , protein kinase a , biology , phosphatidic acid , chemistry , microbiology and biotechnology , protein kinase c , phospholipid , membrane
Phosphatidic acid (PA) phosphatase, which catalyzes the conversion of PA to diacylglycerol, is a key regulatory enzyme in lipid metabolism; it plays a major role in controlling the balance between the synthesis of membrane phospholipids and triacylglycerol. Loss of PA phosphatase in yeast, mouse, and human results in a variety of cellular defects and lipid‐based diseases. The yeast PA phosphatase, Pah1, is regulated by its cellular location (cytosolic versus membrane), which is governed by its phosphorylation and dephosphorylation. The dephosphorylation is catalyzed by the Nem1 (catalytic subunit)‐Spo7 (regulatory subunit) phosphatase complex, and this modification facilitates Pah1 interaction with PA at the membrane for its reaction. Like Pah1, Nem1 and Spo7 are subject to phosphorylation, but the protein kinases involved are largely unknown. In this work, we examined the premise that both subunits are substrates for protein kinase C. Using purified reagents from yeast, we showed that protein kinase C phosphorylated Nem1 or Spo7 on multiple serine residues. Using Nem1 or Spo7 as a substrate, protein kinase C activity was dependent on time, the protein kinase, ATP, and substrate, as well as on phosphatidylserine and diacylglycerol. A multifaceted approach using synthetic peptide substrates, site‐specific mutagenesis, phosphopeptide mapping, and mass spectrometry lead to identification of Ser‐201 and Ser‐210 in Nem1 and Ser‐22 and Ser‐28 in Spo7 as the major sites phosphorylated by protein kinase C. Supported by NIH grant GM05679. Support or Funding Information Supported by NIH grant GM05679. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .