A major enigma in the field cell biology is how signaling proteins with seemingly antithetical functions, such as 1 protein kinase (PK) A and PKC, are regulated temporally and spatially during mitogenic stimulation. Scaffolding proteins are thought to facilitate the efficiency and specificity of substrate phosphorylation and also coordinate the actions of kinases and phosphatases in a spatiotemporal manner. The long-term goal of this research is to understand how SSeCKS, a plasma membrane- and cytoskeleton-associated PKC substrate that binds PKC, PKA, calmodulin and cyclins, helps regulate signaling crosstalk and G1-S progression in untransformed fibroblasts and epithelial cells, compared to prostate cancer cells, in which SSeCKS expression is downregulated severely. Our working hypothesis is that SSeCKS (and its orthologue, GravinfAKAP25O) prevents PKCIPKA crosstalk: in quiescent cells, SSeCKS binds to and inhibits PKC activity whereas during G1-S progression, mitogen-activated phosphorylation of SSeCKS selectively antagonizes PKC binding; PKA, whose binding is phosphorylation insensitive, is then sequestered to """"""""inactive"""""""" sites. With mounting data that SSeCKS has metastasis suppressor activity in prostate cancer, we envision that dissection of the PKC/PKA/SSeCKS control network will further understanding of signaling and cytoskeletal controls during mitogenesis and oncogenesis.
In Aim #1, we will use site-directed mutagenesis and in vitro binding assays to identify the SSeCKS domains that bind PKC and PKA and that mediate multimerization.
In Aim #2, we will examine how SSeCKS binding affects the in vitro kinase activity of PKC and PKA on specific substrates. We will also determine how the binding sites regulate PKC- and PKA-mediated G1-S signaling and cytoskeletal pathways in vivo in untransformed NIH3T3 and prostate epithelial cells, and in SSeCKS-deficient human LNCaP prostate cancer cells. We will analyze how SSeCKS affects the mitogen- and integrin-mediated cellular localization of PKC and PKA.
Aim #3 will investigate how PKC-induced phosphorylation modulates SSeCKS in vivo scaffolding activity and involvement in G1-S regulation. To this end, we will map the in vivo PKC phosphorylation sites on SSeCKS under growth-promoting and oncogenic conditions using a combination of peptide microsequencing and MALDI-TOF mass spectrometry. The role of PKC-induced phosphorylation on SSeCKS scaffolding/regulatory functions will be accessed using SSeCKS phospho-specific antibodies and cell lines expressing SSeCKS PKC-phosphorylation-site mutants. Given that many G1-S regulatory functions are undermined in oncogenesis, the results of the proposed research on SSeCKS should broaden our understanding of spatial and temporal controls governing cell cycle progression in normal and cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA094108-05
Application #
6919212
Study Section
Special Emphasis Panel (ZRG1-PTHC (01))
Program Officer
Ault, Grace S
Project Start
2002-08-01
Project End
2007-07-31
Budget Start
2005-08-10
Budget End
2006-07-31
Support Year
5
Fiscal Year
2005
Total Cost
$289,187
Indirect Cost
Name
Roswell Park Cancer Institute Corp
Department
Type
DUNS #
824771034
City
Buffalo
State
NY
Country
United States
Zip Code
14263
Muramatsu, Masashi; Akakura, Shin; Gao, Lingqiu et al. (2018) SSeCKS/Akap12 suppresses metastatic melanoma lung colonization by attenuating Src-mediated pre-metastatic niche crosstalk. Oncotarget 9:33515-33527
Muramatsu, Masashi; Gao, Lingqiu; Peresie, Jennifer et al. (2017) SSeCKS/AKAP12 scaffolding functions suppress B16F10-induced peritoneal metastasis by attenuating CXCL9/10 secretion by resident fibroblasts. Oncotarget 8:70281-70298
Chattopadhyay, Indranil; Wang, Jianmin; Qin, Maochun et al. (2017) Src promotes castration-recurrent prostate cancer through androgen receptor-dependent canonical and non-canonical transcriptional signatures. Oncotarget 8:10324-10347
Dwyer, Sheila Figel; Gao, Lingqiu; Gelman, Irwin H (2015) Identification of novel focal adhesion kinase substrates: role for FAK in NF?B signaling. Int J Biol Sci 11:404-10
Gelman, Irwin H; Peresie, Jennifer; Eng, Kevin H et al. (2014) Differential requirement for Src family tyrosine kinases in the initiation, progression, and metastasis of prostate cancer. Mol Cancer Res 12:1470-9
Dwyer, Sheila Figel; Gelman, Irwin H (2014) Cross-Phosphorylation and Interaction between Src/FAK and MAPKAP5/PRAK in Early Focal Adhesions Controls Cell Motility. J Cancer Biol Res 2:
Ko, Hyun-Kyung; Akakura, Shin; Peresie, Jennifer et al. (2014) A transgenic mouse model for early prostate metastasis to lymph nodes. Cancer Res 74:945-53
Ko, Hyun-Kyung; Guo, Li-wu; Su, Bing et al. (2014) Suppression of chemotaxis by SSeCKS via scaffolding of phosphoinositol phosphates and the recruitment of the Cdc42 GEF, Frabin, to the leading edge. PLoS One 9:e111534
Cha, Jong-Ho; Wee, Hee-Jun; Seo, Ji Hae et al. (2014) Prompt meningeal reconstruction mediated by oxygen-sensitive AKAP12 scaffolding protein after central nervous system injury. Nat Commun 5:4952
Smeets, Bart; Boor, Peter; Dijkman, Henry et al. (2013) Proximal tubular cells contain a phenotypically distinct, scattered cell population involved in tubular regeneration. J Pathol 229:645-59

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