Difference: RonnieGhose (16 vs. 17)

• Name: Ronnie Ghose
• Email: rghose@sci.ccny.cuny.edu
• Company Name: CUNY CCNY
• Company URL: http://www.ccny.cuny.edu http://www.sci.ccny.cuny.edu/~ghose/welcome.htm
• Location: CUNYCCNYOffice
• Phone: (212) 650-6049 (office), (212) 650-6513 (laboratory)
• Address: Department of Chemistry, The City College of New York 138th Street & Convent Avenue, New York, NY 10031. Office: Marshak Science Building, Room 1314, Laboratory: Marshak Science Building, Room 1324, NMR Laboratory: Marshak Science Building, Room 101
• Country: USA

Grant Number: 1R01GM084278-01A2 Project Title: Catalytic Domain Dynamics in Protein Kinases PI Information: Name Email Title GHOSE, RANAJEET rghose@sci.ccny.cuny.edu

Abstract: DESCRIPTION (provided by applicant): Progression of a host of human cancers is associated with elevated levels of expression and catalytic activity of the Src family of tyrosine kinases (SFKs) making them key therapeutic targets. Even with the availability of multiple crystal structures of active and inactive forms of the SFK catalytic domain, a complete understanding of its catalytic regulation is unavailable. A central step recognized to lead to a dramatic increase in catalytic activity is the phosphorylation of a regulatory tyrosine residue (Tyract) in the "activation loop". This chemical modification is presumed to cause changes in local and long-range interactions and modification of the regulatory dynamics within the catalytic domain. Though some of these changes are inferred from crystal structures, direct evidence is lacking. Solution NMR, the biophysical method best suited to tackle this problem, was previously hindered by difficulties in bacterial expression and purification of sufficient quantities of soluble, properly folded protein for economically viable labeling with NMR-active isotopes. We have through a choice of optimal constructs, co-expression with chaperones and optimization of the purification protocol, achieved the ability to bacterially produce large quantities of the isotopically-labeled catalytic domain of c-Src, the prototypical SFK, and of its Tyract phosphorylated form. This, together with the availability of ultra-high field NMR instrumentation (900 MHz) equipped with the latest generation cryogenic probes and the high-quality of the initial NMR spectra, make the detailed NMR studies of the catalytic domain of the SFKs viable for the first time. We will utilize novel NMR methodology to fully characterize the dynamics of the c-Src catalytic domain, their modifications upon Tyract phosphorylation, their influence on the regulation of enzymatic activity and the mechanism of their perturbation by each of three specific classes of small molecule inhibitors. The SFKs use additional non-catalytic domains to modulate catalytic activity while other protein kinases such as the extracellular signal-regulated kinase (ERK) class of serine/threonine kinases use insertions within the catalytic domain itself in lieu of external domains. Notably, the overall structure and key regulatory elements of the catalytic domain are highly conserved amongst protein kinases. It is thus expected that certain modes of functional dynamics would be conserved while others would vary depending on the class of kinase. We will investigate these effects by ascertaining the functional dynamics in ERK2 (a prototypical ERK), their modification upon dual-phosphorylation of a positive-regulatory activation-loop Thr-X-Tyr motif, for comparison with c-Src. We will also investigate the modifying effects of docking interactions (currently unidentified in SFKs) with regulatory phosphatases, on the functional dynamics in ERK2. Understanding the dynamic underpinnings of kinase activation will likely permit the improvement of current, and the development of new, therapeutic agents for intervention in kinase-associated disorders, especially in cancer and auto-immune diseases. PUBLIC HEALTH RELEVANCE: This project is involved with elucidating the multiple spatial as well as temporal processes involved in the catalytic activation of two key cell signaling molecules namely, c-Src and ERK2. The catalytic activity of these two molecules is tightly regulated in healthy cells. However, this control is lost in a variety of human cancers and proliferative diseases. Thus, a clear understanding of the functioning of these molecules in space and time will improve current anticancer therapies while helping the design of novel strategies targeting this deadly disease.

Public Health Relevance: Project Narrative This project is involved with eluciating the multiple spatial as well as temporal processes involved in the catalytic activation two key cell signaling molecules namely, c-Src and ERK2. The catalytic activity of these two molecules is tightly regulated in healthy cells. However, this control is lost in a variety of human cancers and proliferative diseases. Thus, a clear understanding of the functioning of these molecules in space and time will improve current anticancer therapies while helping the design of novel strategies targeting this deadly disease.

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Institution: CITY COLLEGE OF NEW YORK

1. TH ST AND CONVENT AVE NEW YORK, NY 10031

NSF Org MCB Intial Amendment Date March 5, 2004 Latest Amendment Date March 21, 2005 Award Number 0347100 Award Instrument Continuing grant Program Manager Kamal Shukla MCB Division of Molecular and Cellular Biosciences BIO Directorate for Biological Sciences Start Date March 1, 2004 Expires February 28, 2006 (Estimated) Awarded Amount to Date $175132 Investigator(s) Ranajeet Ghose rghose@sci.ccny.cuny.edu(Principal Investigator) Sponsor CUNY City College Convent Ave at 138th St New York, NY 10031 212/650-5418 NSF Program(s) BIOMOLECULAR SYSTEMS Field Application(s) Program Reference Code(s) BIOT,9251,9183,9178,1164,1045 Program Element Code(s) 1144 Abstract

Abstract

The objective of this project is to develop novel techniques to characterize Nuclear Magnetic Resonance (NMR) relaxation of proteins in the solution state. The development and implementation of these techniques will be facilitated by (i) advances in theoretical methods for experimental design and analysis, (ii) major improvements in commercially available instrumentation, and (iii) the development of cost-effective methods for the bacterial expression and purification of labeled proteins. These methods will be applied to study protein dynamics over a wide range of timescales, with special emphasis on the characterization of protein sidechain dynamics since sidechains play a central role in defining protein-ligand interactions and hence physiological function. These methods will alleviate many of the deficiencies of current experiments by (i) providing information on the details of local dynamics on the fast, ps-ns timescale, (ii) allowing complete characterization of slow, correlated motions on the microsecond-ms timescale, and (iii) being applicable to larger proteins and protein complexes. The PI will utilize molecular dynamics simulations in conjunction with theoretical modeling to identify specific dynamic modes that contribute to the measured relaxation rates. This combination of experimental and computational techniques will allow the identification of key functional modes and the changes thereof on protein interactions. The PI will apply this methodology to elucidate the dynamic determinants of the intra- and inter-molecular interactions involving two cytosolic components of the NADPH oxidase complex - p47phox and p67phox. A very important structural module, the SH3 domain plays a central role in defining protein interactions in these cases. Thus, information gleaned from these studies will provide a self-consistent, space-time view of SH3 domain-mediated protein interactions. The PI will place special emphasis on recruiting students from various backgrounds and at various levels of sophistication, undergraduate and graduate, into his research program. He will redesign undergraduate courses to provide students with the knowledge of cutting edge biophysical techniques and develop a new course on NMR spectroscopy targeted towards advanced undergraduates and graduate students. This course will introduce students to both the theoretical as well as the applied aspects of modern biomolecular NMR spectroscopy both through lectures as well as a series of "hands-on" workshops. The PI currently participates, and will continue to do so, in science outreach programs such as ACS-SEED. This program is designed to facilitate underprivileged high-school students to gain research experience over the summer. CUNY City College has a large minority population. Thus the research and teaching components of this project will encourage those sections of society underrepresented in the sciences to pursue scientific careers both in academia and in industry. Facilitating minority participation in the sciences through teaching, mentoring, career counseling and participating in outreach programs for disadvantaged students is a key goal of this CAREER project.

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