Submitted on February 19, 2003
Accepted on December 19, 2003
SAM68: A DOWNSTREAM TARGET OF ANGIOTENSIN II SIGNALING IN VASCULAR SMOOTH MUSCLE CELLS IN GENETIC HYPERTENSION
Mohammed El Mabrouk1, Quy N. Diep1, Karim Benkirane1, Rhian M. Touyz1, and Ernesto L. Schiffrin1*
1 Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec, Canada
* To whom correspondence should be addressed. E-mail: ernesto.schiffrin{at}ircm.qc.ca.
We investigated whether phosphatidylinositol 3-kinase (PI3K) and SAM68 (Src associated during mitosis, 68 kDa) are involved in angiotensin II (Ang II) growth signaling in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR). PI3K activity was assessed by measuring phosphorylation of the regulatory subunit p85
and kinase activity of the catalytic 110 kDa subunit of PI3K. PI3K/SAM68 interaction was assessed by coimmuniprecipitation and SAM68 activity was evaluated by poly(U) binding. SAM68 expression was manipulated by SAM68 antisense oligonucleotide transfection. VSMC growth was
evaluated by measuring [3H]- leucine and [3H]-thymidine incorporation as indices of protein- and DNA- synthesis respectively. Ang II increased phosphorylation of p85 and kinase activity of the 110 kDa PI3K subunit in VSMCs from SHR and transiently increased p85/SAM68 association. In WKY cells, Ang II increased SAM68 phosphorylation without influencing poly
(U) binding. In SHR Ang II did not influence SAM68 phosphorylation, but increased SAM68 binding to poly (U). Ang II stimulated phosphoinositol phosphate synthesis by PI3K in SAM68 immunoprecipitates in both groups, with significantly enhanced effects in SHR. Inhibition of
PI3K, using the selective inhibitor LY294002, and downregulation of SAM68, by antisense oligonucleotides, significantly decreased Ang II-stimulated incorporation of [3H]-leucine and [3H]-thymidine in VSMCs showing the functional significance of PI3K and SAM68. Our data demonstrate that PI3K and SAM68 are involved in Ang II signalling and that SAM68 is differentially regulated in VSMCs from SHR. These processes may contribute to enhanced Ang II signalling and altered VSMC growth in SHR.
