G protein-coupled receptors (GPCR) agonists, including gastrointestinal (GI) peptides, promote rapid contractile and/or secretory responses in their target cells. The discovery that GI peptides can also induce cell proliferation identified a novel role for these informational molecules. Recent evidence indicates that the mitogenic effects of GPCR agonists are relevant for a number of critical biological processes, including development, compensatory growth and tumorigenesis. The broad, long-term objective of this proposal remains to elucidate the signal transduction pathways that mediate GPCR-induced cell proliferation. Protein kinase C (PKC) occupies a pivotal role in the signal transduction pathways that mediate numerous cellular responses elicited by GI peptides including cell proliferation and modulation of GPCR signaling. However, the events occurring downstream of specific isoforms of PKC remain elusive and our own results demonstrated the existence of PKC-independent pathways leading to mitogenesis. The present challenge is to identify PKC-dependent and PKC-independent downstream targets for GPCRs that transmit signals to the cell interior that participate in the regulation of cell proliferation. Recently, protein kinase D (PKD), cloned in our laboratory, has emerged as a major element in the signal transduction pathways leading to GPCR-induced cell proliferation. Our previous studies demonstrated rapid PKD activation in a broad range of biological systems, including intestinal epithelial cells, via PKC- dependent PKD activation loop phosphorylation on Ser744 and Ser748. In the current proposal, we develop a model that envisages sequential PKD activation involving an early PKC-dependent and a late PKC- independent phase of PKD activation via transphosphorylation and autophosphorylation mechanisms. Additional studies with cells in culture and transgenic mice that express elevated levels of PKD protein in the distal small intestinal and colonic epithelium indicate that PKD plays a major role in mediating proliferation of intestinal epithelial cells, both in vitro and in vivo. Consequently, our central hypothesis is that GPCR agonists activate a PKD-mediated phosphorylation cascade that plays a critical role in the signal transduction of GPCR-induced cellular proliferation. An additional hypothesis is that PKD signaling also operates in the regulation of migration of intestinal epithelial cells. This proposal will examine the following Specific Aims: 1) Characterize early and late phases of PKD activation in response to GPCR activation in intestinal epithelial cells;2) Characterize the role of PKD in the proliferation and migration of intestinal epithelial cells;3) Determine the role of PKD in intestinal cell proliferation in vivo using transgenic expression of PKD. Relevance to Public Health: Gastrointestinal (GI) peptides are chemical messengers that play critical roles in the regulation of cell proliferation and are also implicated in the pathogenesis of important diseases, including chronic inflammatory processes and cancer. In the current application, we propose to continue our studies elucidating the regulation and function of protein kinase D (PKD), a molecule that is emerging as a key player in transmitting GI peptide signals in their target cells. These studies have important implications not only for understanding fundamental regulatory mechanisms in the GI tract but also for identifying PKD as a novel target for therapeutic intervention in diseases characterized by either excessive or defective cell proliferation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK055003-12
Application #
7845556
Study Section
Gastrointestinal Cell and Molecular Biology Study Section (GCMB)
Program Officer
Serrano, Jose
Project Start
1999-03-01
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2011-05-31
Support Year
12
Fiscal Year
2010
Total Cost
$323,978
Indirect Cost
Name
University of California Los Angeles
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Sinnett-Smith, James; Ni, Yang; Wang, Jia et al. (2014) Protein kinase D1 mediates class IIa histone deacetylase phosphorylation and nuclear extrusion in intestinal epithelial cells: role in mitogenic signaling. Am J Physiol Cell Physiol 306:C961-71
Rozengurt, Enrique; Soares, Heloisa P; Sinnet-Smith, James (2014) Suppression of feedback loops mediated by PI3K/mTOR induces multiple overactivation of compensatory pathways: an unintended consequence leading to drug resistance. Mol Cancer Ther 13:2477-88
Ni, Yang; Sinnett-Smith, James; Young, Steven H et al. (2013) PKD1 mediates negative feedback of PI3K/Akt activation in response to G protein-coupled receptors. PLoS One 8:e73149
Yoo, James; Rodriguez Perez, Citlali Ekaterina; Nie, Wenxian et al. (2013) TNF-α and LPA promote synergistic expression of COX-2 in human colonic myofibroblasts: role of LPA-mediated transactivation of upregulated EGFR. BMC Gastroenterol 13:90
Sinnett-Smith, James; Kisfalvi, Krisztina; Kui, Robert et al. (2013) Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: dependence on glucose concentration and role of AMPK. Biochem Biophys Res Commun 430:352-7
Soares, Heloisa P; Ni, Yang; Kisfalvi, Krisztina et al. (2013) Different patterns of Akt and ERK feedback activation in response to rapamycin, active-site mTOR inhibitors and metformin in pancreatic cancer cells. PLoS One 8:e57289
Kisfalvi, Krisztina; Moro, Aune; Sinnett-Smith, James et al. (2013) Metformin inhibits the growth of human pancreatic cancer xenografts. Pancreas 42:781-5
Yoo, James; Rodriguez Perez, Citlali Ekaterina; Nie, Wenxian et al. (2012) TNF-ýý induces upregulation of EGFR expression and signaling in human colonic myofibroblasts. Am J Physiol Gastrointest Liver Physiol 302:G805-14
Waldron, Richard T; Innamorati, Giulio; Torres-Marquez, M Eugenia et al. (2012) Differential PKC-dependent and -independent PKD activation by G protein α subunits of the Gq family: selective stimulation of PKD Ser⁷⁴⁸ autophosphorylation by Gαq. Cell Signal 24:914-21
Young, Steven H; Rozengurt, Nora; Sinnett-Smith, James et al. (2012) Rapid protein kinase D1 signaling promotes migration of intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 303:G356-66

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