Cid755673 Enhances Mitogenic Signaling by Bombesin and EGF Through a Protein Kinase D-Independent Pathway

EUGENIA TORRES-MARQUEZ; JAMES SINNETT-SMITH; SUSHOVAN GUHA; OSVALDO REY; RICHARD WALDRON; ENRIQUE ROZENGURT

Congreso; Digestive Diseases Week; 2010

AGA

Background: Protein kinase D1 (PKD1) is the founding member of a new protein kinase family, separate from the previously identified PKCs. PKD1 is rapidly activated by G protein- coupled receptor (GPCR) agonists, including gastrointestinal regulatory peptides, via a protein kinase C (PKC)-dependent and independent pathways. PKD1 has been implicated in the regulation of multiple processes, including DNA synthesis and cell proliferation induced by GPCR agonists. Consequently, the development of specific, cell-permeable PKD family inhibitors would be extremely useful in helping to identify the physiological substrates and define the biological roles of the PKD family as well as for developing novel therapeutic approaches in a variety of pathological conditions. Results: Recently, CID755673 was reported to act as a potent and highly selective inhibitor of PKD family members (Sharlow, E. R. et.al. J. Biol. Chem. 283, 33516-33526, 2008). In the course of experiments using CID755673, we noticed that this compound exerted unexpected and potent stimulatory effects on [3H]thymi- dine incorporation and cell cycle progression in Swiss 3T3 stimulated by gastrointestinal peptides and growth factors. Specifically, exposure to CID755673 (5-50 ⎧M) further enhanced [3H]thymidine incorporation induced by the Gq-coupled receptor agonist bom- besin or the biologically active phorbol ester PDBu in control or PKD-expressing Swiss 3T3 cells. In view of the established role of PKD1 signaling in DNA synthesis in response to bombesin or phorbol esters established by our laboratory, the results obtained with CID755673 were surprising since rather than abrogate DNA synthesis, this compound produced a further stimulation of [3H]thymidine incorporation in different derivatives of Swiss 3T3 cells. Indeed, siRNAs targeting PKD1 depleted PKD1 protein (~90%) but did not prevent the increase in DNA synthesis induced by CID755673. Conclusion: We conclude that CID755673 induces cellular responses through molecular targets other than PKD1. While the identification of the putative target(s) of CID755673 that mediates its growth- stimulatory effects is of interest, the major point raised by our study is that CID755673 can not be considered a specific inhibitor of PKD and it should be used with extreme caution in experiments attempting to elucidate the role of the PKD family in any cellular process, particularly cell proliferation.