Tyrosine phosphorylation of the alpha subunit of transducin and its association with Src in photoreceptor rod outer segments.
Recent evidence indicates that tyrosine phosphorylation may play important roles in retinal photoreceptor rod outer segments (ROS). We investigated the tyrosine phosphorylation of endogenous proteins in isolated bovine ROS. Several proteins with apparent molecular masses of 31, 39, 60, 83, 90, 97, 120, 140, and 180 kDa were tyrosine-phosphorylated in ROS incubated with Mg(2+), ATP, and orthovanadate. Several tyrosine kinase inhibitors significantly inhibited tyrosine phosphorylation of these proteins in ROS. The 39- and 60-kDa tyrosine-phosphorylated proteins were identified as the alpha subunit of the G protein transducin (Talpha) and the tyrosine kinase Src, respectively. The presence of Src and tyrosine kinase activity in bovine ROS was confirmed by their cofractionation with rhodopsin and Talpha on continuous sucrose gradients. Several tyrosine-phosphorylated proteins, including Src, coimmunoprecipitated with Talpha. The association of Src with Talpha was detected in the absence of tyrosine phosphorylation, but was enhanced with increased tyrosine phosphorylation of ROS. Moreover, tyrosine kinase activity also associated with Talpha was sevenfold higher under tyrosine-phosphorylating conditions. The recovery of transducin by hypotonic GTP extraction from tyrosine-phosphorylated ROS was significantly less than that from nonphosphorylated ROS. We localized the site on Talpha phosphorylated by Src to the amino-terminal half by limited tryptic digests, and further mapped it by ion trap mass spectrometry to Tyr(142) in the helical domain of Talpha. Talpha was also tyrosine-phosphorylated in vivo in rat retina, but this phosphorylation was not affected by light.
Bell
M W
MW
Department of Biochemistry, Baylor College of Medicine, Houston, Texas, USA.
Desai
N
N
Guo
X X
XX
Ghalayini
A J
AJ
D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D012160:Retina / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D015293:Transducin / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D000936:Antigen-Antibody Complex; D005136:Eye Proteins; D010446:Peptide Fragments; D014443:Tyrosine; D006160:Guanosine Triphosphate; D019061:src-Family Kinases; D014357:Trypsin; D015293:Transducin