Identification of Nck interacting proteins in vascular smooth muscle cells

The adaptor molecule Nck has been demonstrated to mediate Angiotensin II (AngII)-induced stimulation of p21-activated kinase (PAK) and c-Jun NH2-terminal kinase (JNK) in vascular smooth muscle cells (VSMC). We have previously demonstrated, that immunoprecipitation of Nck from VSMC stimulated by AngII yielded an unidentified 100 kD phosphotyrosine (pTyr) protein. The present study was aimed at identifying the Nck-associated 100 kD pTyr protein in VSMC. Several candidate proteins of appropriate size, that had been shown previously either to bind to Nck or had been implicated in signal transduction pathways leading to activation of PAK or JNK were tested for association with Nck in VSMC. The first candidate protein we tested was Git1, which did not bind to Nck in VSMC upon stimulation by AngII. However, we identified dynamin as a 100 kD protein that was bound to Nck in VSCM via interaction with the third Nck-SH3 domain. However, dynamin was not tyrosine phosphorylated by AngII treatment and seemed to be distinct from the 100 kD phosphotyrosine protein that was found in Nck immunoprecipitates. Future work will now have to identify the Nck-associated 100 kD pTyr protein and functional studies will have to address its role in AngII signaling.