Abstract B01: Phosphorylation of pro-oncogenic c-Src at serine-17 by protein kinase G-Iα promotes chemoresistance/cell proliferation in human non-small cell lung cancer cells.

We previously showed that PKG-Iα promotes cell survival in neural cells (N1E-115 neuroblastoma and NG108-15 neuroblastoma-glioma hybrid cells) and significantly contributes to serine-155 phosphorylation of BAD, an apoptosis-regulating protein (Johlfs and Fiscus 2010). We also found, by using both gene knockdown and pharmacological inhibitors (ODQ, DT-2 and DT-3) to decrease PKG-Iα kinase activity, that PKG-Iα promotes cell proliferation and chemoresistance in human ovarian cancer cells, involving a novel interaction between PKG-Iα and the oncogenic protein c-Src (Fiscus et al 2012, Leung et al 2010). PKG-Iα activity was dependent on c-Src-catalyzed tyrosine phosphorylation of PKG-Iα; and, reciprocally, c-Src activation was dependent on expression and kinase activity of PKG-Iα;. We hypothesized that activation of c-Src involves PKG-Iα-catalyzed phosphorylation at serine-17 of c-Src, because amino acids around serine-17 provide consensus sequence for PKG-I, and tαhat this plays an important role in proliferation and chemoresistance. As c-Src has been proposed to promote cell proliferation and chemoresistance in NSCLC cells, the present study aimed to: 1) determine if c-Src increases PKG-Iα kinase activity in NSCLC cells using our newly-developed near-infrared-fluorescence (NIRF)-kinase assay, 2) determine if serine-17-phosphorylation of c-Src, in intact cancer cells, is dependent on PKG-Iα expression and kinase activity (using siRNA gene knockdown and pharmacological inhibitors), and 3) determine if inhibition/knockdown of c-Src and PKG-Iα alters chemoresistance and proliferation of NSCLC cells. Protein expression of c-Src and PKG-Iα was determined by conventional Western blot analysis and new ultrasensitive capillary-electrophoresis-based NanoPro100/1000 (ProteinSimple, Santa Clara, CA, USA). NanoPro100/1000 allows clear separation and identification of different c-Src and PKG-Iαphospho-forms (a novel way of identifying the activation of c-Src and PKG-Iα). We found PKG-Iα was able to directly catalyze phosphorylation of serine-17 of c-Src, increasing autophosphorylation of c-Src at tyrosine-419, the activation site of c-Src. Inhibition of PKG-Iα kinase activity using DT-2 or silencing of PKG-Iα expression using siRNA dramatically reduced the intracellular phosphorylation of c-Src at serine-17. Both kinase-activity inhibition and knockdown of PKG-Iα caused significant increases in apoptosis, synergistically enhancing apoptosis induced by the chemotherapeutic agent cisplatin, and dramatically decreased cell proliferation/colony formation in NSCLC cells. This novel reciprocal relationship between c-Src and PKG-Iα provides a new target for the development of new anti-cancer therapeutic agents. Fiscus RR, Leung EL, Wong JC, Johlfs MG (2012). Nitric oxide/protein kinase G-Ialpha promote c-Src activation, proliferation and chemoresistance in ovarian cancer. In: Farghaly S (ed). Ovarian Cancer - Basic Science Perspective. Intech Open Access Publisher. pp 315-334. Johlfs MG, Fiscus RR (2010). Protein kinase G type-Ialpha phosphorylates the apoptosis-regulating protein Bad at serine 155 and protects against apoptosis in N1E-115 cells. Neurochem Int 56: 546-553. Leung EL, Wong JC, Johlfs MG, Tsang BK, Fiscus RR (2010). Protein kinase G type Ialpha activity in human ovarian cancer cells significantly contributes to enhanced Src activation and DNA synthesis/cell proliferation. Mol Cancer Res 8: 578-591. Citation Format: Mary G. Johlfs, Ronald R. Fiscus, Priyatham Gorjala, Renee Coffman, Harry Rosenberg. Phosphorylation of pro-oncogenic c-Src at serine-17 by protein kinase G-Iα promotes chemoresistance/cell proliferation in human non-small cell lung cancer cells. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B01.