Many signaling proteins are recruited or regulated by the D-3 phosphoinositides (PI (3,4)P2 and PI(3,4,5)P3. These D-3 phosphoinositides serve as membrane localization elements that recruit different signaling enzymes to specific locations on the plasma membrane. The target proteins contain unique D-3 phosphoinositide-recognition motifs that serve as the primary signal transducers enabling the protein to be recruited or regulated by these lipid second messengers to initiate downstream signaling. Since these phosphoinositides represent a minor component of the phospholipids, the question is how is their binding specificity achieved. This research is aimed at determining how they specifically bind to their target proteins. In the first project src homology 2 (SH2) domains and pleckstrin homology (PH) domains will be used to investigate the structural basis for phosphoinositide binding specificity. Based on previous work on the SH2 domains of the p85 regulatory subunit of PI 3-kinase, Dr. Chen has proposed consensus sequences for PI(3,4,5)P3 and PI(3,4)P2 binding. In this study other SH2 and PH domains will be examined. Different strategies including site-directed mutagenesis will be used to test his working model for the binding selectivity. Moreover a combinatorial peptide library approach will be employed to identify structural factors that control the binding affinity. Since the sequence analysis suggests that calmodulin-binding motifs on some regulatory proteins share homology with the PI(3,4,5)P3 or PI(3,4)P2 binding sequence, a hypothesis is proposed that these phosphoinositides may exert enzyme regulation through the control of calmodulin (CaM) availability to cellular targets. During cell activation, D-3 phosphoinositides may increase CaM availability by sequestering CaM-binding proteins. Thus the binding profiles of these CaM binding peptide fragments will be examined, as well as other experiments to test this. Since phospholipid-protein interactions are a driving force for transmembrane signal transduction, understanding the molecular basis of this recognition will aid in the elucidation of the regulatory mechanisms of many cellular functions.
