Identification of potential substrates of protein kinase A by comparative analysis of proteins immunoenriched with phospho-specific antibodies in Yarrowia lipolytica

LUDOVICO, IVO DÍAZ ; GONZALEZ, MARÍA CECILIA; GIACOMETTI, ROMINA; KRONBERG, MARÍA FLORENCIA

Mar del Plata

Congreso; LI Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2015

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The cAMP-dependent protein kinase (PKA) is an enzyme that engages the transference of a phosphate group of ATP to its target protein; its activity is regulated by intracellular cAMP concentration. In Yarrowia lipolytica, a dimorphic fungus of biotechnological interest, we found that PKA is involved in morphogenesis, cell metabolism and adaptation to stress conditions. The regulatory and catalytic subunits are encoded by sole genes, RKA1 and TPK1, respectively. In this work different proteomics and molecular biology techniques were adapted to identify potential target proteins of Y. lipolytica PKA. The strategy used was based on comparison of immunoenriched proteins with an anti-phosphoPKA substrate antibody from a wild type strain and a mutant strain without PKA activity (Δtpk1). The protocol performed involved the linkage of protein A-Sepharose to a monoclonal anti-phosphoPKA substrate antibody. Phosphoproteins from the wild type and mutant strains were enriched, and were resolved by 2D-PAGE electrophoresis. Finally, spots only found in the wild type strain and absent in the PKA mutant were selected and the proteins were identified by MALDI-TOF. It was possible to identify three putative PKA substrate proteins: the self regulatory subunit of PKA, Rka1; a protein with homology to Saccharomyces cerevisiae ubiquinone biosynthesis monooxygenase CoQ6 and the translational elongation factor EF1-α.