Abstract 215: Characterization by mass spectrometry of protein kinase C substrates differentially phosphorylated in LNCaP cells in response to phorbol ester and bryostatin 1 treatment
Authors: Noemi Kedei, Sudipto Das, Thorkell Andresson, Peter M. Blumberg
Journal: Cancer Research (2017)

Abstract Bryostatin 1 (bryo 1) is a natural product of therapeutic interest for cancer and Alzheimer disease. Its unique behavior as a protein kinase C activator that paradoxically antagonizes many but not all phorbol ester responses has led to intense interest in its mechanism of action. Recently, using microarray analysis in two different cellular systems (LNCaP prostate cancer and U937 leukemia cell lines) where the typical phorbol ester PMA and bryo 1 have different biology, we have shown that a core mechanism contributing to the unique biology of bryo 1 is transiency of action resulting in a variable extent of decreased or missing late responses. We have excluded that there is a class of genes whose transcription is uniquely regulated at early times by bryo 1. In continuing to explore the mechanisms underlying the variable transiency of the responses by bryo 1 we have evaluated the substrates phosphorylated after treatment with PMA and bryo 1. Since PMA and bryo 1 induce differential subcellular localization of PKCs, they should result in differential access to substrates and consequent differences in the pattern of substrate phosphorylation. Whole cell lysates of LNCaP cells treated for 30 min with fully effective doses (100 nM) of PMA and bryo 1 and with DMSO as vehicle control were subjected to mass spectroscopic analysis. The comprehensive analysis identified several thousand phosphopeptides after drug treatment including the expected phosphopeptides for ERK2 (S185, Y187) and PKCdelta (S299, S302, S304). Many of peptides were similarly phosphorylated in response to both drugs (e.g. S641/S646 of Fam129B, S310 of actin-related protein 2/3 complex subunit 1B, S876 of RhoGEF and pleckstrin domain-containing protein 2, or multiple previously unknown sites for PKD1 (S239, S247), while a limited number were differentially phosphorylated. PMA specific phosphorylations included PKCdelta at Y313, mTOR at T2471, MAP4 at S624 and/or T627, E3 ubiquitin-protein ligases ZNRF2 at S82 and HUWE1 at S3818. Bryo 1 specific phosphorylations included BCL2-like 13 at S303, TOM1L2 at S424, and MAP2K2 in the T17, T25, S26 region. Selected specific phosphorylations are being validated using phostag gels, a method that enables the separation of phosphorylated proteins from their non-phosphorylated counterparts on SDS-gels. The identification of PKC substrates that are differentially phosphorylated by bryo 1 should both facilitate screening of other ligands capturing the biological behavior of bryo 1 as well as further illuminate the specifics of the pathways downstream of PKC activation by these differently acting ligands. Citation Format: Noemi Kedei, Sudipto Das, Thorkell Andresson, Peter M. Blumberg. Characterization by mass spectrometry of protein kinase C substrates differentially phosphorylated in LNCaP cells in response to phorbol ester and bryostatin 1 treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 215. doi:10.1158/1538-7445.AM2017-215