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cPKC isozyme specific substrates in murine embryonic stem cells.
Author(s) -
Costa Helio Miranda,
Andrade Alexander,
Labate Carlos,
Krieger José Eduardo,
MochlyRosen Daria,
Schechtman Deborah
Publication year - 2008
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.22.1_supplement.1050.8
Subject(s) - protein kinase c , phosphorylation , isozyme , activator (genetics) , peptide , microbiology and biotechnology , embryonic stem cell , biology , kinase , chemistry , biochemistry , enzyme , gene
PKCs are intracellular signalling enzymes that participate in embryonic stem cells (ES) biology and most likely modify targets associated with key processes including self‐renewal and differentiation. We first identified the PKC isozymes that are present in ES and used an exploratory approach to select potential substrates using cell based assays and phosphoproteomic. Murine ES express mainly PKCs α, βI, βII, δ, ε and ζ/λ. PMA or cPKC peptide modulators were used to identify cPKC substrates, which were resolved in 2D gels and developed with phospho‐specific dye. Upon treatment with cPKC‐selective activator peptide, ψβRACK, 41‐unique protein spots were identified, 9 increased phosphorylation. In contrast, treatment with the cPKC inhibitor, βC2‐4, resulted in a decreased phosphorylation of 51 spots and disappearance of 6. Furthermore, 27 spots disappeared upon treatment with βIPKC‐selective inhibitor, βIV5‐3, and 11 had a decrease in phosphorylation, whereas 10 spots disappeared with βIIPKC‐specific inhibitor, βIIV5‐3, and 11 ‐ decreased phosphorylation. Nine spots increased their intensity with the cPKC activator and inhibited with either beta PKC=specific inhibitor peptide and 8 spots were inhibited by more than one inhibitor peptide. Preliminary data using MS/MS revealed an increased phosphorylation of splicing factor 1 following treatment with either PMA or cPKC activator peptide while elongation factor 1 δ increased phosphorylation with PMA, but decreased with cPKC inhibitor treatment. Taken together, we began the identification of substrates of specific PKC isozymes in mES using isozyme‐selective inhibitors and activators, which deserve to be investigated in the future.