Arthur M. Edelman - US grants

The identification of myosin and its discrete localization in neural tissues has stimulated research focused on a wide variety of possible functions for this protein including growth cone motility, exocytosis, dendritic shape changes and modulation of post- synaptic surface morphology. Neuronal myosin like other nonmuscle myosins may require phosphorylation of its light chain subunits in order to interact with actin and thus generate force. The initial phosphorylation event is therefore critically important in the regulation of the functions which myosin may subserve in neural tissue. We and others have identified several calcium- regulated protein kinases potentially capable of phosphorylating brain myosin, namely type II Ca2+, calmodulin-dependent kinase and myosin light chain kinase (MLCK). In order to explore this regulatory system we propose: 1) to detrmine the immunocytochemical localization of MLCK in the central nervous system, in cultured sympathetic neurons and in isolated brain subcellular fractions, 2) to study the sites and kinetics of the phosphorylation of brain myosin by brain MLCK and type II Ca2+, calmodulin-dependent kinase and 3) to study myosin phosphorylation and its cytoskeletal association in intact nerve endings as functions of depolarization and calcium influx. The results of this research will yield important information about a critical regulatory step in a number of fundamental functions of the neuronal cytoskeleton.

DESCRIPTION: Extracellular signal-control elevations in intracellular Ca2+ levels regulate a diverse array of cellular phenomena. Evidence has accumulated for calcium dyshomeostatsis in nervous system pathologies including HIV-induced neuronal injury, Alzheimer's disease and Parkinson's disease suggesting the potential for new pharmacological interventions for these disorders based on alteration in intracellular Ca2+ levels. Calcium calmodulin (CaM) dependent protein kinases (CaM kinase) are important mediators of the effects of elevated levels of intracellular calcium upon physiologically relevant target proteins. CaM kinase I is the focus of this proposal. This research is intended to provide an integrated approach for elucidating the signaling cascade for phosphorylation and activation of CaM kinase I. The specific aims are: 1. Identification of extracellular and intracellular signals which induce phosphorylation and activation of CaM kinase I in pheochromocytoma PC12 cells; 2, Analysis of signal transduction mechanisms in the CaM kinase I cascade. Specifically to determine by immunoprecipitation techniques if CaM kinase I (or CaM kinase IV) forms complexes with its activating kinases (CaMKIKs) and evaluate whether phosphorylation of CaMKIKs influence their abilities to phosphorylate and activate CaM kinase I and to form complexes with CaM kinase I.; 3. Evaluation of the respective roles of CaMKIKs in CaM kinase cascades. Specifically to determine through studies of their respective kinetics of phosphorylation whether CaMKIKs utilize CaM kinase I preferentially as substrate relative to CaM kinase IV and determine their respective localization using Northern and immunoblotting and immunocytochemistry, and whether the CaMkKIKs have cell type or tissue specific functions.

[unreadable] DESCRIPTION (provided by applicant): Protein phosphorylation is an essential and pervasive mechanism for the regulation of cellular and organismal function in nature. However for many individual phosphorylation events occurring in vivo, the technical difficulty of simultaneously identifying both the particular protein phosphorylated and the phosphorylating kinase has been a major impediment to understanding their unique physiological roles. This project will design, and optimize the technical aspects of, a method to screen for the intracellular substrates of protein kinases. This method is anticipated to have broad applicability to the study of cellular signaling processes. The principle of the method is to first map by 2-D gel electrophoresis the pattern of proteins phosphorylated in vivo in response to stimuli known to activate a particular class of protein kinases; and then to identify proteins, the stimuli-specific phosphorylation of which, is diminished by intracellular depletion of a particular kinase within that class. The Specific Aims are: Specific Aim 1 To document the presence and develop methods for relative quantification of, the kinases to be tested, CaMKIalpha and CaMKIV in a neuroblastoma cell line (BE(2)C) and in cultured primary cortical neurons. Specific Aim 2 To design small interfering RNAs (siRNAs) and test their effectiveness in knocking down individual CaMKs in these cells. Specific Aim 3 To map the patterns by 2-D gel electrophoresis of protein phosphorylation in these cells by in vivo 32p-labeling and/or fluorescent phosphoprotein staining and evaluate these patterns in response to agonists which elevate the concentration of intracellular calcium (Ca2+i). Specific Aim 4 To identify by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/MS) and peptide mass fingerprint database search, proteins, the Ca2+i-dependent phosphorylation of which is inhibited by specific CaMK knockdowns. [unreadable] [unreadable]