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Reconstitution and biochemical characterization of WAVE regulatory complex

The Wiskott Aldrich syndrome protein family of proteins (WASP, N‐WASP, WAVE 1–3), are regulators of actin dynamics. While WASP regulation by GTPases and kinases is well characterized both biochemically and structurally, little is known about WAVE regulation. WAVE exists as part of a complex of five proteins termed the WAVE Regulatory Complex (WRC). It consists of WAVE, Sra , Nap, Abi and HSPC300. Biochemical studies of WRC have been hampered by the difficulty of expressing WRC components in bacterial, insect or yeast expression systems. We have modified pFastBac vector by adding the lobster myosin promotor region, which has been previously shown to increase the expression level of proteins in insect cells. Baculoviruses yielding high expression of each component of WRC were obtained. Co‐infection has allowed efficient expression and purification of WRC and two sub‐complexes, Sra‐Nap and WAVE‐Abi‐HSPC300. Preliminary data show that WRC is active in actin assembly assays and that Rac GTPase, a previously reported potential activator, does not further activate it. Further, we find that only WAVE is phosphorylated when WRC is incubated with Abl kinase, which has been reported to interact with Abi component of WRC. Further experiments with the purified WRC and its sub‐complexes will shed light on the mechanism of WRC regulation.