Tyrosine Phosphorylation of the α Subunit of Transducin and Its Association with Src in Photoreceptor Rod Outer Segments

• Published in: Journal of neurochemistry Vol. 75; no. 5; pp. 2006 - 2019
• Main Authors: Bell, Michael W., Desai, Nirav, Guo, Xiao X., Ghalayini, Abboud J.
• Format: Journal Article
• Language: English
• Published: Oxford UK Blackwell Science Ltd 01-11-2000; Blackwell
• Subjects: Animals; Antigen-Antibody Complex - chemistry; Binding Sites; Biological and medical sciences; Cattle; Cell Membrane - metabolism; Cell physiology; Eye Proteins - metabolism; Fundamental and applied biological sciences. Psychology; G proteins; Guanosine Triphosphate - metabolism; Molecular and cellular biology; Peptide Fragments - analysis; Phosphorylation - drug effects; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Rats; Retina; Retina - metabolism; Rod Cell Outer Segment - metabolism; Rod outer segments; Sequence Analysis, Protein; Spectrometry, Mass, Electrospray Ionization; Src; src-Family Kinases - antagonists & inhibitors; src-Family Kinases - chemistry; src-Family Kinases - metabolism; Transducin; Transducin - metabolism; Trypsin - metabolism; Tyrosine - metabolism; Tyrosine phosphorylation; Vision, photoreception; Tyrosine; Phosphorylation; Rod; Photoreceptor; Transducin; Retina; Ox; Eye; Visual system; Signal transduction; Vertebrata; Mammalia; Outer segment; Aminoacid; Alpha-Peptide chain; Artiodactyla; Ungulata; G protein
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.2000.0752006.x

: 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 Mg2+, 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 α subunit of the G protein transducin (Tα) 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 Tα
on continuous sucrose gradients. Several tyrosine‐phosphorylated proteins,
including Src, coimmunoprecipitated with Tα. The association of Src with
Tα was detected in the absence of tyrosine phosphorylation, but was
enhanced with increased tyrosine phosphorylation of ROS. Moreover, tyrosine
kinase activity also associated with Tα 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 Tα
phosphorylated by Src to the amino‐terminal half by limited tryptic digests,
and further mapped it by ion trap mass spectrometry to Tyr142 in the helical domain of Tα. Tα was also tyrosine‐phosphorylated in vivo in rat retina, but this phosphorylation was not affected by light.