Specific detection of phosphatidylinositol 3,4,5-trisphosphate binding proteins by the PIP3 analogue beads: an application for rapid purification of the PIP3 binding proteins.
Phosphatidylinositol (PI) 3-kinase is known as one of the key molecules involved in the various biological events such as vesicle trafficking, cytoskeletal rearrangements and cell survival. T clarify the molecular basis underlying these events, we have tried to identify the proteins that can interact with phosphatidylinositol 3,4,5-trisphosphate (PIP3), the lipid product of PI3-kinase. Using a new PIP3 analogue, PIP3-APB, we synthesized an affinity column for PIP3 binding proteins. This enabled us to purify and identify several PIP3 binding proteins such as Tec tyrosine kinase, Gap1m, and Akt, as the candidates for the downstream molecules of PI3-kinase. All of these proteins contain PH domains, possible binding sites for phospholipids. Studies with various deletion mutants of Tec or Gap1m revealed that their PH domains are indeed the binding sites for PIP3. These results demonstrate that this PIP3-analogue binds various PIP3 binding proteins with high specificity and may be useful to elucidate the downstream mechanisms of PI3-kinases-mediated signaling pathways.
Shirai
T
T
Department of Applied Biological Chemistry, Graduate School of Agriculture and Life Science, University of Tokyo, Japan.
Tanaka
K
K
Terada
Y
Y
Sawada
T
T
Shirai
R
R
Hashimoto
Y
Y
Nagata
S
S
Iwamatsu
A
A
Okawa
K
K
Li
S
S
Hattori
S
S
Mano
H
H
Fukui
Y
Y
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D001921:Brain; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D008863:Microspheres; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000138:chemical synthesis / Q000378:metabolism; D010750:Phosphoproteins; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013950:Thymus Gland; D020703:ras GTPase-Activating Proteins
D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; D002352:Carrier Proteins; C112403:PIP3-APB; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D011506:Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C039077:platelet protein P47; D020703:ras GTPase-Activating Proteins; C089848:Tec protein-tyrosine kinase; D011505:Protein-Tyrosine Kinases