8 細胞内情報伝達機構の解明を指向したPIP_3結合型アフィニティゲルの作製とその結合タンパクの探索(口頭発表の部)

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Phosphatidylinositol 3,4,5-trisphosphate (PIP_3: 1) is a polyphosphoinositide produced from phosphatidylinositol 4,5-bisphosphate (PI4, 5P_2) by phosphatidylinositol-3 kinase (PI-3 kinase) in response to a wide range of growth factor stimulation. It is still unclear what a important role of PIP_3 is. To investigate how PIP_3 interact with proteins in the intracellular signal transduction system, we have developed two affinity gels with novel PIP_3 analogs. IP_3-APBgel is a affinity gel bearing 1-O-acyl type ligand (IP_3-APB: 6) and is expected to associate with proteins which specifically recognize head group of PIP_3. Using this IP_3-APBgel, we have isolated a novel protein with a molecular mass of 43kD from bovine brain extract and was termed PIP_3 binding protein. The protein bound PIP_3 with a higher affinity than it did inositol 1,3,4,5-tetrakisphosphate (IP_4), PI4, 5P_2, phosphatidylinositol 3,4-bisphosphate (PI3, 4P_2) and phosphatidylinositol 3-phosphate (PI3P), suggesting that the binding to PIP_3 was specific. It contained one zinc finger motif and two pleckstrin homology (PH) domains and the entire sequence was 83% similar to centaurin α, another PIP_3 binding protein. The binding activity was weaker in the mutants with a point mutation in the conserved sequences in each PH domains and the introduction of both mutations caused loss of the binding activity. These suggest PIP_3BP binds PIP_3 throuth two PH domains present in the molecule. To detect PIP_3 binding proteins in a more exact manner, we designed PIP_3 analog containing a glycerol group as well as fatty acid groups (PIP_3-APB: 12). One of the peptides identified from the lysates of calf thymus had a molecular mass of 70kD and was identified to be the Tec protein-tyrosine kinase. This interaction between the PIP_3 analog beads and Tec could be efficiently inhibited by free PIP_3. Significantly higher concentrations of PI4, 5P_2, PI4P and IP_4 were required to compete this interaction. Studies with the various deletion mutants of Tec protein revealed that the binding site of PIP_3 was localized within the PH domain of Tec. Now we have demonstrated that our affinity gels are very useful biochemical tools to detect PIP_3 associated proteins. Moreover, we isolated a novel PIP_3 binding protein, Tee and Aktγ (which was also isolated by PIP_3-APBgel). These proteins have PH domains which turned out to be essential to interact with PIP_3. This PIP_3-PH interaction is likely to have a significance in the PI-3 kinase signal transdution pathway.
天然有機化合物討論会の論文
1997-07-20

8 細胞内情報伝達機構の解明を指向したPIP_3結合型アフィニティゲルの作製とその結合タンパクの探索(口頭発表の部)

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