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Eukaryotic and Prokaryotic Protein Kinases


Protein kinases transfer the γ-phosphate of ATP to the hydroxyl groups of specific serine, threonine (serine/threonine kinases) or tyrosine(tyrosine kinases) residues in substrate proteins. This covalent modification of the Ser/Thr/Tyr–OH moiety is a fundamental mechanism of cellular regulation and intracellular signal transduction and has a significant role in almost every aspect of cell growth, differentiation, maturation, motility and regulated cell death in eukaryotes. Tyrosine phosphorylation, long recognized as a central signaling mechanism in higher eukaryotes, is beginning to emerge as an important mechanism in prokaryotic signal transduction and has been shown to play a central role in bacterial pathogenesis. Towards our long term goal of assessing the functional implications of the transfer of a phosphate from ATP to specific amino-acid residues on key protein targets across the three domains of life, we use biophysical and biochemical tools to obtain structure-function relationships in a variety of protein kinases.

Eukaryotic elongation factor 2 kinase


The activity of the GTPase, eukaryotic elongation factor 2 (eEF-2) facilitates the translocation of the nascent polypeptide chain from the ribosomal A-site to the P-site during protein synthesis. Phosphorylation of eEF-2 (on Thr-56) decreases its affinity for the ribosome and leads to a global reduction in translational elongation rates. Eukaryotic elongation factor 2 kinase (eEF-2K), a member of the α-kinase family, that is ubiquitously expressed in eukaryotes, enables this critical post-translational modification. eEF-2K is activated through a unique calmodulin-mediated mechanism that is regulated by phosphorylation, calcium ions and pH. Our goal is decipher the mechanisms involved in the activation and activity of eEF-2K.

Bacterial tyrosine kinases


The BY-kinases (for bacterial tyrosine kinases) comprise the largest family of protein tyrosine kinases in the bacterial taxa and are highly conserved in both Gram-positive and Gram-negative species. BY-kinases have been shown to participate in a variety of cellular processes including the synthesis and export of polysaccharides involved in the formation of biofilms or capsules. Instead of the dual-lobed structure characteristic of the eukaryotic protein kinases, the catalytic domains of BY-kinases resemble P-loop NTPases. Our goal is to understand the mechanisms through which the BY-kinases have repurposed teh ancient P-loop fold to evolve the ability to phosphorylate on tyrosine.

Mitogen activated protein kinases


The mitogen activated protein kinase (MAPK) ERK2 (extracellular signal regulated kinase 2) lies at the terminus of a three-tiered phosphorylation-based response to a wide range of extracellular cues that include cytokines, hormones and growth factors. ERK2 phosphorylates a large number of substrates both in the nucleus and in the cytoplasm, including a variety of transcription factors, regulatory kinases, phosphatases, proteins of the nuclear pore complex, cytoskeleton proteins, among others. ERK2-mediated phosphorylation occurs on specific serine/threonine residues that immediately precede a proline. However, the (S/T)P motif alone does not provide sufficient substrate affinity or selectivity to distinguish it from other proline-directed kinases. In order to attain a high level of specificity for its native substrates, ERK2 and other MAP kinases utilize one of two so-called “docking sites”, named the D-recruitment site (DRS) and the F-recruitment site (FRS), that are spatially distinct from the catalytic site. Our goal is to understand how these docking interactions support specific phosphorylation events within MAP kinase substrates.