c-src (c-Src), 1 a nonreceptor protein-tyrosine kinase, is distributed widely in various cell types and is potentially involved in the signal delivery for controlling cellular growth and function (1, 2). In the resting fibroblasts, the activity of c-Src kinase is tightly controlled by phosphorylation, and the enzyme is found in an inactivated state in which the carboxyl-terminal tyrosine 527 (Tyr-527) is phosphorylated to high stoichiometry (3-5). The Tyr-527 phosphorylation is done by Csk, another non-receptor type protein-tyrosine kinase (6, 7), and dephosphorylation by phosphotyrosine phosphatase, which causes the activation of c-Src kinase (2, 3). On the other hand, v-Src kinase, which lacks a tail sequence containing Tyr-527, is not regulated by the phosphorylation. This is why uncontrolled and constitutively activated v-Src kinase plays a major role for unlimited cell growth (1-4). Recently, tertiary structures of c-Src kinase and related molecules were defined (8 -10), and dephosphorylation of phospho-Tyr-527 or binding of viral Nef protein with the SH3 domain has been suggested to destabilize the whole kinase structure for activation. It is still an open question whether some other chemical events could also destabilize the structure of the Src kinase for activation.Nitric oxide (NO), which is synthesized enzymatically from L-arginine and molecular oxygen by nitric-oxide synthases (11) or NO-generating chemicals have been shown to affect a number of biological systems regulating various physiological and biochemical functions (12). Some of these include reduction of protein kinase C activity (13) and activation of p21 ras (14). Lander et al. (15) report that treatment of lymphoma cells with NO-generating agents increased the catalytic activity of p56 lck kinase, possibly through potentiating the phosphotyrosine phosphatase activity. The present study, to our knowledge, provides for the first time evidences that NO released from these chemicals activates Src kinase through a Tyr-527-independent and Tyr-416-linked mechanism, which involves S-nitrosylation/S-S bond-mediated modification of Src molecules.
EXPERIMENTAL PROCEDURESCells and Chemicals-The murine NIH3T3 fibroblast cell line overexpressing c-Src kinase was kindly provided by Dr. D. Shalloway of Pennsylvania State University. v-Src-transformed NIH3T3 cells were from our own stock. The cell lines were cultured in plastic plates with Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37°C in a 5% CO 2 , 95% air incubator. After becoming confluent, cells were collected with 0.25% trypsin, 0.01% EDTA in phosphatebuffered saline and were split into 60-mm plastic plates with Dulbecco's modified Eagle's medium containing 10% fetal calf serum for a further 20 -24-h incubation. The cells were then rinsed with fresh modified Eagle's medium twice and incubated in modified Eagle's medium at 37°C for 1 h before use. S-Nitroso-N-acetyl penicillamine (SNAP) was purchased from Research Biochemicals Int. (Natick, MA), and sodium nitroprus...