Guo X X, Huang Z, Bell M W, Chen H, Anderson R E
Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Mol Vis. 2000 Nov 4;6:216-21.
We have previously shown that phosphatidylinositol 3-kinase (PI 3-kinase) activity is present in bovine rod outer segments (ROS). The present study was undertaken to investigate the mechanism of PI 3-kinase activation in these membranes.
Tyrosine-phosphorylated ROS (PY-ROS) were obtained by incubating ROS with ATP, MgCl2, and orthovanadate (Na3VO4), a tyrosine phosphatase inhibitor. Non-phosphorylated ROS (N-ROS) were obtained by incubating ROS under the same conditions, but without ATP and orthovanadate. Both were subjected to immunoprecipitation using antibodies against the regulatory p85 (anti-p85) subunit of PI 3-kinase, the catalytic p110 (anti-p110) subunit of PI 3-kinase, or phosphotyrosine (anti-PY). The immunoprecipitates (IPs) were assayed for PI 3-kinase activity. Enzyme assay products were separated by thin-layer chromatography (TLC), deacylated, and identified by high performance liquid chromatography (HPLC).
PI 3-kinase activity in anti-p85 and p110alpha IPs was significantly higher in PY-ROS than in N-ROS. No enzyme activity was recovered in anti-p110beta IPs. PI 3-kinase activity in anti-PY IPs from PY-ROS was six-fold greater than those from N-ROS. Immunoblot analysis showed that the amount of p85 in PY IPs from PY-ROS was significantly higher than those from N-ROS. However, tyrosine phosphorylation of p85 and p110alpha was not observed in anti-p85 and anti-p110alpha IPs that were probed with anti-PY.
This study indicates that the p85/p110alpha complex of PI 3-kinase is present in ROS and tyrosine phosphorylation is involved in the regulation of its activity.
我们之前已经表明磷脂酰肌醇3激酶（PI 3激酶）活性存在于牛视杆细胞外段（ROS）中。本研究旨在探讨这些膜中PI 3激酶激活的机制。
通过将ROS与ATP、MgCl2和酪氨酸磷酸酶抑制剂原钒酸钠（Na3VO4）一起孵育获得酪氨酸磷酸化的ROS（PY-ROS）。通过在相同条件下但不添加ATP和原钒酸钠孵育ROS获得非磷酸化的ROS（N-ROS）。两者都使用针对PI 3激酶调节性p85（抗p85）亚基、PI 3激酶催化性p110（抗p110）亚基或磷酸酪氨酸（抗PY）的抗体进行免疫沉淀。对免疫沉淀物（IPs）进行PI 3激酶活性测定。酶测定产物通过薄层色谱（TLC）分离、脱酰基，并通过高效液相色谱（HPLC）鉴定。
PY-ROS中抗p85和p110α IPs中的PI 3激酶活性显著高于N-ROS中的。在抗p110β IPs中未检测到酶活性。PY-ROS的抗PY IPs中的PI 3激酶活性比N-ROS的高六倍。免疫印迹分析表明，PY-ROS的PY IPs中p85的量显著高于N-ROS的。然而，在用抗PY探测的抗p85和抗p110α IPs中未观察到p85和p110α的酪氨酸磷酸化。
本研究表明PI 3激酶的p85/p110α复合物存在于ROS中，酪氨酸磷酸化参与其活性调节。
