| Cellular Signaling involves orchestrated subcellular targeting of peripheral proteins. The primary objective of this thesis is to understand the molecular mechanisms of membrane targeting and activation of different families of signaling proteins, including phospholipase C-delta (PLC-delta), protein kinase C (PKC) and phospholipase D (PLD). This study particularly focuses on elucidating the interactions of the individual membrane targeting domains namely the C1, C2 and PX domains with the membranes. We use multi-disciplinary approaches that involve a wide variety of physical, chemical and biological techniques.; PLC-delta belong to the superfamily of lipolytic enzymes that catalyze the hydrolysis of phosphatidylinositol 4, 5 bisphosphate (PI(4,5)P2) to inositol 1,4,5 trisphosphate (IP3) and 1,2-diacylglycerol (DAG). PLC-delta consists of a catalytic domain and three regulatory domains: PH, EF-hand, and C2 domains. To understand the role of the C2 domain, we measured the binding of the C2 domains of PLC-delta1, delta3, delta4 to model membranes, and analyzed in terms of their electrostatic properties. Results establish that the C2 domains of PLC-delta isoforms are Ca2+-dependent and have distinct membrane binding properties, which in turn control their subcellular localization behaviors.; PLD is another class of lipases which catalyze the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. Apart from the catalytic domain, PLD1 isoform has PH and PX as its membrane targeting domains. This study was undertaken to determine the membrane targeting mechanism of the PX domain, with an emphasis on determining the phosphoinositide (PI) specificities. Results from in vitro membrane binding measurements provide new insight into the membrane preference of PX domain.; PKC are a family of serine/threonine kinases that mediate numerous cellular processes. All PKCs contain an amino-terminal regulatory domain and a carboxyl terminal catalytic domain. Regulatory domains contain tandem C1 domains and a C2 domain. The C1 domain is the interaction site for DAG and phorbol ester. However, correlation between their intrinsic DAG affinities and the relative importance in PKC activation is unclear. Our studies with isolated C1 domains and the full-length PKC-alpha and PKC-gamma proteins unravel the molecular basis for differential membrane targeting and activation mechanisms of these two closely related isoforms. |
