The broad, long-term objective of this proposal is to understand at the molecular level the mechanism by which phosphoinositide 3-kinase (PI 3- K) activates cellular signaling. PI 3-K is implicated in diverse cellular functions and deregulation of its biological signal results in the transformation of cells. Evidence is emerging that the lipid products of PI 3-K, PtdIns-3,4-P2 and PtdIns-3,4,5-P3 have multiple effectors in the cell. Presumably agonist-stimulated accumulation of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 on the inner leaflet of the plasma membrane recruits cytosolic proteins for activation. My published and preliminary studies suggest that PI 3-K lipid products can activate the protein kinase C (PKC) isoforms PKCepsilon and PKCzetu in vitro. Preliminary studies presented in this proposal also show that cells expressing an active PI 3-K and producing PtdIns-3,4-P2 and PtdIns-3,4,5-P3 have elevated PKCepsilon and PKCzetu activity. I also show that the protein kinase Akt/PKB associates with and activates PKCzetu in vivo. Finally, a direct binding of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 to PKC's is demonstrated. In the proposal, experiments are designed to determine the role of PtdIns-3,4,5-P3, Akt/PKB and PDK-1 in the regulation of the PKCepsilon, zetu and mu in vitro and in vivo. Activation of PKC's in vitro will be assessed using synthetic D3 phosphoinositides in the presence of Akt/PKB or PDK-1. Mutant PKC's will be made to demonstrate that PDK-1 phosphorylates a critical threonine residue, leading to activation. Mutant PKC's will be made in which the regulatory regions of PKCepsilon and PKCalpha will be switched to test the specificity of activation. Activation of PKC's in vivo will be investigated using constitutively active PI 3-K, Akt/PKB and PDK-1 mutants. Association of PKC's with Akt/PKB will also be investigated. The binding of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 to PKC's will also be measured. Finally, the lipid- binding site on PKC's will be determined using a labeled photoactivatable PtdIns-3,4,5-P3 which can be cross-linked to target proteins. The results of these studies will elucidate the role of PI 3-K products in cellular signaling. A hypothesis is proposed in which multiple mechanisms exist to regulate PKC's in the PI 3-K signaling pathway, including lipid binding, phosphorylation and association with other proteins. A better understanding of PI 3-K signaling with emphasis on its lipid products has implications for the understanding of cell function and may provide novel therapeutic approach for the treatment of human disease.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
7R01CA075134-02
Application #
6124561
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Program Officer
Spalholz, Barbara A
Project Start
1998-12-15
Project End
2003-11-30
Budget Start
1999-12-01
Budget End
2000-11-30
Support Year
2
Fiscal Year
2000
Total Cost
$291,069
Indirect Cost
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02215
Christoforides, Claudine; Rainero, Elena; Brown, Kristin K et al. (2012) PKD controls ýývýý3 integrin recycling and tumor cell invasive migration through its substrate Rabaptin-5. Dev Cell 23:560-72
Nhek, Sokha; Ngo, Mike; Yang, Xuemei et al. (2010) Regulation of oxysterol-binding protein Golgi localization through protein kinase D-mediated phosphorylation. Mol Biol Cell 21:2327-37
Storz, Peter; Döppler, Heike; Copland, John A et al. (2009) FOXO3a promotes tumor cell invasion through the induction of matrix metalloproteinases. Mol Cell Biol 29:4906-17
Watkins, Janis L; Lewandowski, Katherine T; Meek, Sarah E M et al. (2008) Phosphorylation of the Par-1 polarity kinase by protein kinase D regulates 14-3-3 binding and membrane association. Proc Natl Acad Sci U S A 105:18378-83
Kunkel, Maya T; Toker, Alex; Tsien, Roger Y et al. (2007) Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter. J Biol Chem 282:6733-42
Storz, Peter; Doppler, Heike; Ferran, Christiane et al. (2005) Functional dichotomy of A20 in apoptotic and necrotic cell death. Biochem J 387:47-55
Storz, Peter; Doppler, Heike; Toker, Alex (2005) Protein kinase D mediates mitochondrion-to-nucleus signaling and detoxification from mitochondrial reactive oxygen species. Mol Cell Biol 25:8520-30
Doppler, Heike; Storz, Peter; Li, Jing et al. (2005) A phosphorylation state-specific antibody recognizes Hsp27, a novel substrate of protein kinase D. J Biol Chem 280:15013-9
Hausser, Angelika; Storz, Peter; Martens, Susanne et al. (2005) Protein kinase D regulates vesicular transport by phosphorylating and activating phosphatidylinositol-4 kinase IIIbeta at the Golgi complex. Nat Cell Biol 7:880-6
Storz, Peter; Doppler, Heike; Toker, Alex (2004) Protein kinase Cdelta selectively regulates protein kinase D-dependent activation of NF-kappaB in oxidative stress signaling. Mol Cell Biol 24:2614-26

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