POWRE: Determination of the Catalytic Activity and Functional Requirement for a Novel Kinase Homology Domain at the C-terminus of Newly Cloned Putative Calcium Channel

Harvard University, Cambridge MA

Investigators

Abstract

The ability of a cell to respond to its environment is one of the most basic of functions and requires a network of intracellular molecules that work in concert to integrate a response. Of the molecules involved in signal transduction, calcium is the most fundamental, serving as a universal second messenger in a plethora of pathways. Many of the effects of calcium are mediated by calcium/calmodulin (Ca2+/CaM) regulated protein kinases. Amongst such kinases, elongation factor-2 kinase (eEF-2K), which controls protein translation by phosphorylating elongation factor-2, stands alone. It has a catalytic structure which is distinct from any other protein serine/threonine/tyrosine kinase or histidine kinase family member. The functional significance of this different kinase structure is unknown, although it is clear that eEF-2K has a non-redundant cellular function, underscoring the importance of its catalytic domain. Another kinase from the amoebae Dictyostelium, which phosphorylates myosin heavy chain on specific threonine residues, shares this unique catalytic domain This kinase also appears to have a nonredundant cellular function since mutagenesis of its target threonine residues in myosin heavy chain results in gross overassembly of myosin and causes defects in myosin-related contractile processes (Egelhoff T. T. et al., Cell., 75:363, 1995). In order to explore the functional diversity of this new kinase family, a novel gene was cloned from an EST that encodes an eEF-2K-related C-terminal kinase domain. This new sequence, named elongation factor-2 kinase related channel (EFKRC), not only shares homology to the unique catalytic structure of eEF-2K, but is part of a larger protein that also has strong homology to a novel putative calcium channel, melastatin (MLSN1). Importantly, EFKRC is the first protein found to contain an eEF-2K catalytic homology that serves as a domain within a much larger protein. In addition, since eEF-2K is a Ca2+/CaM regulated kinase, the potential exists for the EFKRC kinase domain to play a fundamental functional role within the context of this putative calcium channel. The goal of this project is to determine if the kinase domain (KD) of EFKRC is a catalytically active kinase and/or is required for EFKRC function. This will be accomplished by determining if the kinase domain of EFKRC is active when expressed individually and/or in the context of the EFKRC full-length molecule both in in vitro kinase assays and in in vivo cellular 32 P-orthophosphate labeling experiments. In addition, the catalytic function of EFKRC will be assessed by determining its ability to substitute for the catalytic core of eEF-2K with the generation and analysis of chimeric molecules and point mutants. The functional requirement of the KD of EFKRC will be determined by analyzing the ability of full-length (FL) and KD-null EFKRC to complement an EFKRC-dependent lethality found in DT40 chicken B cells. In addition, bulk calcium assays will be performed that will compare the effects of FL EFKRC vs. KD-null EFKRC. Overall, these studies should provide critical insights into the functionality of the EFKRC kinase domain. It is unknown why nature utilizes an unusual kinase domain structure for some essential threonine phoshorylation events. During evolution, this domain was incorporated into at least one larger protein, EFKRC. This project will yield new insights into whether larger proteins that incorporate this unique kinase family domain also utilize it in a non-redundant, essential fashion. This POWRE award will allow Dr. Nadler, who is entering a new research area, to gather sufficient preliminary data in order to submit a regular proposal.