Abstract 3195: Protein kinase Cβ substrate screens reveal enrichment for mRNA binding proteins and identify SERBP1 as a novel PKC substrate and signal transduction hub protein.
Authors: O'Brien Peter J. , Kang Xiaolin , Zachwieja Joseph , Lippincott John 
Published: August 2014

PKCβ is implicated in carcinogenesis and tumor progression, potentially via its influence on protein translation. Relatively few PKCβ substrates have been identified, however, limiting our understanding of these processes. Here, we use high throughput kinase screens and gene ontology (GO) annotation enrichment analysis to identify putative PKC substrates and potentially related biological themes. Protein microarrays spotted with 4085 Saccharomyces cerevisiae proteins and 2754 human proteins were probed with a PKCβ catalytic domain in the presence of γ33 ATP, lipid activators and divalent calcium. Three concentrations of the kinase spanning a 50-fold concentration range were compared to protein kinase A- and kinase-free control reactions. 117 yeast proteins and 112 human proteins yielded PKCβ phosphorylation Z scores greater than 3.0, including the known PKC substrates Annexin 2 and CPI-17/PPP1R14a. 53 human proteins and 41 yeast proteins that were dose-proportionally phosphorylated by PKCβ were considered high confidence PKCβ substrates. Over 90% of these are known polyanion binding proteins (PABP) suggesting that protein charge distribution may influence PKCβ-substrate interactions. PKCβ phosphorylated WD repeat proteins, DEAD box helicases, Eukaryotic Initiation Factors, La-related proteins, and Casein Kinase 2 family paralogs on both arrays. GO analysis of all human high confidence hits showed greater than six fold enrichment in the terms “RNA binding” and “ribonucleoprotein complex” (Benjamini P-value &lt;0.0008 and 0.0006, respectively). One high confidence hit, the PAI-1 mRNA binding protein SERBP1, is structurally related to the signaling hub protein and PKC substrate Ki1/57. A modulator of PAI-1 mRNA stability, SERBP1 is a highly connected hub protein that has been linked to tumor status and cancer progression. Here, site-directed mutagenesis and phospho-specific antibodies were used to show that recombinant PKCβ uniquely phosphorylates SERBP1 serine 74 in a highly conserved amino terminal motif distinct from the PKC motif in Ki1/57. SERBP1 phosphorylation was also demonstrated in leukemia cells and peripheral blood cells treated with phorbol esters, a reaction that was blocked in a dose-dependent manner by isoform-selective PKC inhibitors. These findings suggest that mRNA binding proteins and other PABP represent a new class of PKCβ substrates, and that some of the known effects of PKC on protein translation may arise through direct interactions with mRNA binding proteins.

Citation Format: Peter J. O'Brien, Xiaolin Kang, Joseph Zachwieja, John Lippincott. Protein kinase Cβ substrate screens reveal enrichment for mRNA binding proteins and identify SERBP1 as a novel PKC substrate and signal transduction hub protein. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3195. doi:10.1158/1538-7445.AM2013-3195