Assays for Chemotaxis and Chemoattractant-Stimulated TorC2 Activation and PKB Substrate Phosphorylat

Chemotaxis is a highly coordinated biological system where chemoattractants trigger multiple signal transduction pathways which act in concert to bring about directed migration. A signaling pathway acting through PIP3 , which accumulates at the leading edge of the cell, has been extensively characterized. However, chemotaxis still remains in cells depleted of PIP3 , suggesting there are PIP3 -independent pathways. We have identified a pathway involving TorC2-PKBR1 as well as another containing PLA2 activity that act in parallel with PIP3 . Activation of PKBR1, a myristoylated Protein Kinase B homolog, is dependent on TorC2 (Rapamycin-insensitive Tor complex 2) kinase but is completely independent of PIP3 . In response to chemoattractant, PKBs rapidly phosphorylate at least eight proteins, including Talin B, PI4P 5-kinase, two RasGefs, and a RhoGap. These studies help to link the signaling pathways to specific effectors and provide a more complete understanding of chemotaxis.