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.

Lysophosphatidic acid stimulates p21-activated kinase in vascular smooth muscle cells.

Lysophosphatidic acid (LPA) has been shown to be a potent mitogen for vascular smooth muscle cells. Src-dependent transactivation of receptor tyrosine kinases has been previously demonstrated to mediate LPA-induced activation of MAP kinase ERK1/2. Furthermore, generation of reactive oxygen species (ROS) by LPA is also known to contribute to MAP kinase activation. Rho family small G-proteins Rac and Cdc42, and their immediate downstream effector p21-activated kinase (PAK), have been demonstrated to mediate important effects on the cytoskeleton that are relevant for cell migration and proliferation. In the present report we evaluated stimulation of PAK by LPA in rat aortic vascular smooth muscle cells (VSMC) by PAK immunocomplex MBP in-gel kinase assay. LPA increased PAK activity 3-fold, peaking at 5 min and showing sustained activation up to 45 min. Inhibition of tyrosine kinases by pretreatment of VSMC with genistein or specific inhibition of Src by PP1 greatly diminished LPA-induced PAK activation, whereas specific inhibition of PDFG- and EGF receptor kinase by tyrphostin AG1296 and AG1478 had no effect. Furthermore, inhibition of Galpha(i) by pertussis toxin and inhibition of NADH/NADPH oxidase by diphenylene iodonium also diminished LPA-induced stimulation of PAK. This is the first study to demonstrate that LPA activates PAK. In VSMC, PAK activation by LPA is mediated by Galpha(i) and is dependent on Src, whereas EGF- or PDGF receptor transactivation are not involved. Furthermore, generation of ROS is required for LPA-induced activation of PAK.