Free Radical Research Center

Andrey Sorokin, PhD

Description Of Research Interests:

Laboratory is interested in developing an understanding of the details of signaling pathways leading from G-protein coupled receptors and tyrosine kinases through adaptor proteins to downstream signaling molecules. The differential stimulation and abortion of intracellular signaling pathways set the stage for the wide variety of cellular responses and determines in part the specificity of signaling by G-protein coupled receptors and tyrosine kinases. Research in this laboratory is primarily focused on characterizing the molecular mechanisms underlying the activation and termination of signaling pathways, as well as defining the cellular consequences of specific stimulation of these cascades in systems relevant for the signaling from G-protein coupled receptors. The other major area of research is the role of cyclooxygenase-2 and prostaglandins in the control of resistance of cells to apoptosis. By combining of molecular biological, biochemical and cellular biological approaches the defining the alterations in signal transduction which lead to pathological phenotype can be obtained.
 

Three major projects in the laboratory are:

I. Endothelin-induced activation of protein kinases and small GTP-binding proteins
Endothelin-1 (ET-1) is a member of a family of 21-amino-acid vasoactive and proliferative peptides that exerts a wide variety of biological activities. ET-1 interacts with a specific, G-protein-coupled, seven transmembrane domain receptor on the cell membrane. Upon binding to its receptor ET-1 elicits a series of short-term transmembrane signals, which, in glomerular mesangial cells (GMC), include activation of different phospholipases, mobilization of Ca2+, activation of protein kinase C, as well as expression of the early response genes. ET-1-dependent contraction of GMC can regulate the glomerular ultrafiltration coefficient and ET-1 induced glomerular hypertrophy and proliferation may contribute to diverse types of proliferative and sclerotic glomerular diseases.

In particular, we are interested in 1) identifying mechanisms involved in regulation of signaling via guanine nucleotide exchange factor Pix; 2) evaluation of the role of cytoplasmic tyrosine kinase Pyk2; 3) elucidation of the negative regulation of endothelin signaling on the level of intracellular MAPK cascades by dual specificity phosphatases.

II. Role of adaptor proteins Crk and p66 Shc in Kidney Pathology
The common theme of adaptor proteins is that they serve to bring two or more proteins together by utilizing one or more of many different binding domains, whilst having no enzymatic properties themselves. During the last several years we have gained insight into the mechanism of regulation of cellular signaling via adaptor protein Crk. We have begun to define the role of Crk in regulation of distinct intracellular signaling cascades and have cloned a novel and biologically distinct member of the Crk family, that we have called CrkIII. Adaptor protein p66 Shc controls oxidative stress apoptotic responses and regulates life span in mammals. The ability of p66 Shc to control oxidative cell response apoptosis and animal longevity is unequivocally dependent on Ser phosphorylation of p66 Shc. We have shown that endothelin-1 (ET-1) induces p66 Shc serine phosphorylation in glomerular mesangial cells, resulting in it's association with adaptor protein 14-3-3.

Based on findings obtained during the past several years, the working hypothesis upon which experimental protocols of this project are designed is: that signaling via Crk and p66 Shc is extremely important for the proliferation of glomerular mesangial cells and their resistance to oxidative stress apoptotic agents and is affected during the progression of kidney diseases.

III. Cyclooxygenase 2 and apoptosis
The principal target of non-steroidal anti-inflammatory drugs is cyclooxygenase, the enzyme which catalyses the first, rate-limiting step in the formation of prostaglandins from arachidonic acid. Two discrete cyclooxygenase gene products, termed COX-1 (constitutively expressed in most cell types) and COX-2 (expressed after stimulation by growth factors and cytokines) have been cloned and characterized. COX-1 and COX-2 play important roles in different physiological situations and disease processes ranging from inflammation to cancer. The role of COX-2 in the cell has been the focus of intense investigation since discovery of this enzyme. We have recently demonstrated the anti-apoptotic effect of COX-2 in trophic withdrawal apoptosis in PC-12 cells (5) and provided evidence that COX-2 promotes cell survival by a mechanism linking increased expression of pro-survival genes coupled to inhibition of NO- and superoxide-mediated apoptosis. A schematic representation of the proposed model of Cox-2-dependent apoptosis regulation is presented (Fig. 1).

Figure 1. Hypothetical model for the regulation of cell survival by Cox-2. Stimulation of Cox-2 expression by agonists is associated with increased production of PGE2 from arachidonic acid (AA). PGE2 is released from cells and can stimulate the prostaglandin receptors (EP) on GMC, leading to increased expression of proteins that decrease the production and availability of nitric oxide (NO) and in parallel, oppose pro-apoptotic pathways independent of NO, such as Bim signaling. Cox-2 expression also increases expression of multidrug resistance P-glycoprotein (MDR1), which regulates efflux of apoptotic compounds (X) from GMC promoting cell survival. In vivo this leads to increased number of mesangial cells, mesangial expansion and may contribute to glomerular disease secondary to inflammation, hypertension and diabetes.

Selected Recent Publications:

1. Foschi M., Chari, S., Dunn, M.J., and Sorokin, A. Biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase. EMBO J. 16:6439-6451, 1997.
2. Sorokin A., Reed, E., Nkemere N., Dulin N.O., and Schlessinger J., Crk Protein Binds to PDGF Receptor and Insulin Receptor Substrate-1 with Different Modulating Effects on PDGF- and Insulin-dependent Signaling Pathways. Oncogene. 16:2425-2434, 1998.
3. Sorokin A., and Reed A. Insulin stimulates the tyrosine dephosphorylation of docking protein p130cas (Crk-associated substrate), promoting the switch of the adaptor protein Crk from p130cas to newly phosphorylated insulin receptor substrate-1. Biochemical J. 334:595-600, 1998.
4. Dulin, N.O., Sorokin, A., Reed, E., Elliott, S., Kehrl, J.H., and Dunn, M.J. RGS3 inhibits G protein - mediated signaling via translocation to the membrane and binding to Ga11. Mol. Cell. Biol. 19:714-723, 1999. 
5. McGinty A., Chang Y.-W. Sorokin A., Bokemeyer D., and Dunn M.J. Cyclooxygenase II expression inhibits trophic withdrawal apoptosis in nerve growth factor differentiated PC12 cells. J. Biol. Chem. 275:12095-12101, 2000. 
6. Chang, Y.W., Jakobi, R. McGinty, A., Foschi, M., Dunn, M.J. and Sorokin, A. Cycooxygenase-2 promotes cell survival by stimulation of Dynein Light Chain Expression and inhibition of neuronal nitric oxide synthase activity. Molec. Cell. Biol. 20:8571-8579, 2000.
7. McGinty, A., Foschi, M., Chang, Y.W., Han, J., Dunn, M.J. and Sorokin, A. Induction of Prostaglandin Synthase-2 by Mitogen-Activated Protein Kinase Cascades. Biochem. J. 352:419-424, 2000.
8. Sorokin, A., Kozlowski, P., Graves, L., and Philip, A. Protein tyrosine kinase Pyk2 mediates endothelin-induced p38 MAP kinase activation in glomerular mesangial cells. J. Biol. Chem. 276:21521-21528, 2001.
9. Foschi, M., Franchi, F., Han, J., La Villa, G., and Sorokin A. Endothelin-1 Induces Serine Phosphorylation of the adaptor protein p66Shc and It's Association with 14-3-3 Protein In Glomerular Mesangial Cells. J. Biol. Chem. 276:26640-26647, 2001.
10. Patel, V., Dunn, M. and Sorokin, A. Regulation of MDR-1 (P-glycoprotein) by cyclooxygenase-2. J. Biol. Chem. 277:38915-38920, 2002.
11. Miller, B., Chang Y.-W.E. and Sorokin A. Cyclooxygenase 2 Inhibits SAPK activation In neuronal apoptosis. Biochem. Biophys. Res. Comm. 300:884-888, 2003.
12. Ishaque, A., Dunn, M. and Sorokin, A. Cyclooxygenase-2 inhibits TNFalpha-mediated apoptosis in renal glomerular mesangial cells. J. Biol. Chem. 278: 10629-10640, 2003.
13. Prosser, S., Sorokina, E., Pratt, P., H. and Sorokin, A. CrkIII: A novel and biologically distinct member of the Crk family of adaptor proteins. Oncogene. 22:4799-4806, 2003.
14. Sorokin, A., and Kohan, D. Physiology and pathology of endothelin-1 in the renal mesangium. AJP: Renal Physiology. 285:F579-589, 2003.
15. Pratt, P.P., Bokemeyer, D., Foschi, M., Sorokin, A., and Dunn, M.J. Alterations In subcellular localization of p38 MAPK potentiate endothelin-stimulated COX-2 expression In glomerular mesangial cells. J. Biol. Chem. 278: 51928-51936, 2003.
16. Sorokin, A. Cyclooxygenase-2: potential role In regulation of drug efflux and multiudrug resistance phenotype. Curr. Pharm. Des. 10:647-657, 2004.
17. Chahdi A, Sorokin A, Dunn MJ, Landry Y.  The Rac/Cdc42 guanine nucleotide exchange factor beta1Pix enhances mastoparan-activated Gi-dependent pathway in mast cells.
    Biochem Biophys Res Commun. 317: 384-9, 2004.
18. Chahdi A, Miller B, Sorokin A. Endothelin 1 induces beta 1Pix translocation and Cdc42 activation via protein kinase A-dependent pathway. J Biol Chem. 280: 578-84, 2005.