University of Virginia Health System

Signal Transduction through Protein Kinase C

The major interest of my laboratory is in activation of Protein Kinase C (PKC) isozymes and in roles of these enzymes in cellular signaling networks. PKC family members are activated by association with cell membranes and help to transduce a variety of extracellular signals. Most cells have several PKC isozymes. Alterations in isozyme expression or activation have been found in a number of diseases including some cancers and some endocrine, immunological, neurological, and cardiovascular diseases. Our goal is to understand the structure and activation of the enzymes and the role of individual isozymes in specific cellular processes. This understanding should facilitate design of more specific PKC activators and inhibitors for clinical use.

Properties of membrane lipids that activate PKC are studied using biochemical and biophysical techniques. Structural analysis of PKC is conducted in collaboration with Dr. Kretsinger (Biology) via generation of 2-dimensional crystals on defined lipid monolayers and with Drs. Grisham and Cafiso (Chemistry) using NMR and EPR. In a past collaboration with Drs. Steers and Tuttle (Urology), we started to examine the hypothesis that PKC may transduce physical stimuli such as cell stretch or pressure changes that occur in the urinary tract or the vasculature.

Among the proteins regulated by PKC are many ion channels and receptors that also are affected by anesthetics. We hypothesize that some anesthetic effects may be mediated via alterations in PKC activation, either via direct anesthetic-PKC binding or indirectly via anesthetic effects on membrane lipid domains that activate PKC. In collaboration with Drs. Lynch, Kamatchi, Patel, Zuo and Bayliss (Anesthesiology), we are analyzing the regulation by PKC of calcium, sodium, and potassium channels and of gluatamate transporters involved in anesthetic actions. In collaboration with Dr. Hussaini (Pathology), we have studied the role of PKC isozymes in development of invasive glioblastomas. A newer project also involving collaborations with Drs. Gaylinn (Endocrinology), Stevenson and Somlyo (Physiology) addresses the regulation by PKC of a peptide hormone that has effects on several ion channels and on lymphocyte, cardiovascular, adipocyte, and osteoblast function.

GRANT SUPPORT

Sando % effort - 20% 3 RO1 GM31184-17S1 Sando (PI) 9-30-03 – 8-30-07, NIH/NIGMS Structure and Activation of Protein kinase C isozymes. The aims of this grant are I) to determine the structures of PKC isozymes on a lipid surface in the presence of various activators, inhibitors and substrates using 2D, crystallography and II) to determine whether lipid modulators affect PKC by altering domain formation in the membrane in vitro.

1 RO1 CA90851-01 Hussaini (PI) 7-01-02 – 6-30-07, Sando % effort - 10 % NIH/NCI The role of PKCh in regulating astrocytoma invasive growth. The specific aims of this grant are to determine I) whether PKCh expression or activation differs between neoplastic and non-neoplastic human astrocytes, II) how PKCh expression is controlled in astrocytic tumors, and III) whether manipulation of PKCh expression alters growth, migration/invasion or apoptotic properties of neoplastic astorcytes.

1 RO1 GM065211-01A Zuo (PI) 2-01-03 – 1-31-08, Sando % effort - 12 %NIH/NIGMS Volatile anesthetic modulation of glutamate transporters. The specific aims are to determine I) whether volatile anesthetics affect trafficking, phosphorylation and activity of glutamate transporters, and II) whether volatile anesthetics reduce ischemia/hypoxia-induced reversed transport of glutamate.

1 RO1 GM65214-01A Kamatchi (PI) 8-01-03 – 7-30-08, Sando % effort - 15% NIH/NIGMS PKC modulation of calcium current and anesthetic action. The aims are I) to identify PKC-induced phosphorylation sies in the a 1 subunits of Cav channels in Xenopus oocytes expressing Ca v channels and muscarinic M1 or angiotensin AT 1A receptors, II) to identify PKC isozymes involved in the actions of each agonist, and III) to identify the PKC isozymes through which volatile anesthetics affect the Ca v channels

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