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.
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.
|Piserchio, Andrea; Warthaka, Mangalika; Kaoud, Tamer S et al. (2017) Local destabilization, rigid body, and fuzzy docking facilitate the phosphorylation of the transcription factor Ets-1 by the mitogen-activated protein kinase ERK2. Proc Natl Acad Sci U S A 114:E6287-E6296|
|Lee, Kwangwoon; Alphonse, Sébastien; Piserchio, Andrea et al. (2016) Structural Basis for the Recognition of Eukaryotic Elongation Factor 2 Kinase by Calmodulin. Structure 24:1441-51|
|Will, Nathan; Piserchio, Andrea; Snyder, Isaac et al. (2016) Structure of the C-Terminal Helical Repeat Domain of Eukaryotic Elongation Factor 2 Kinase. Biochemistry 55:5377-86|
|Piserchio, Andrea; Ramakrishan, Venkatesh; Wang, Hsin et al. (2015) Structural and Dynamic Features of F-recruitment Site Driven Substrate Phosphorylation by ERK2. Sci Rep 5:11127|
|Temel, Deniz B; Dutta, Kaushik; Ghose, Ranajeet (2014) Sequence-specific backbone ¹H, ¹³C and ¹?N assignments of the catalytic domain of the Escherichia coli protein tyrosine kinase, Wzc. Biomol NMR Assign 8:37-41|
|Temel, Deniz B; Dutta, Kaushik; Alphonse, Sébastien et al. (2013) Regulatory interactions between a bacterial tyrosine kinase and its cognate phosphatase. J Biol Chem 288:15212-28|
|Ghose, Ranajeet (2013) A measured approach: determining the PLC?1 docking site on Itk using a biochemical ruler. J Mol Biol 425:679-82|
|Piserchio, Andrea; Francis, Dana M; Koveal, Dorothy et al. (2012) Docking interactions of hematopoietic tyrosine phosphatase with MAP kinases ERK2 and p38?. Biochemistry 51:8047-9|
|Piserchio, Andrea; Dalby, Kevin N; Ghose, Ranajeet (2012) Assignment of backbone resonances in a eukaryotic protein kinase - ERK2 as a representative example. Methods Mol Biol 831:359-68|
|Piserchio, Andrea; Cowburn, David; Ghose, Ranajeet (2012) Expression and purification of Src-family kinases for solution NMR studies. Methods Mol Biol 831:111-31|
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