|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 2004 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 2003 : ¥2,200,000 (Direct Cost : ¥2,200,000)
Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express DOCK5, a putative Rac guanine nucleotide exchanger, as a CrkL-binding protein. We cloned DOCK5 and raised specific antibodies against the molecule. We overexpressed CrkL, DOCK5, or both in combination in COS7 cells. Overexpressed DOCK5 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both DOCK5 accumulated at CrkL-induced focal adhesions, suggesting a functional implication of the interaction.
Using specific antibodies against isoforms of WAVE (WASP [Wiskott-Aldrich syndrome protein] family Verprolin-homologous protein, also called Scar), we demonstrated that human platelets express all 3 isoforms. With the use of an in vitro pull-down technique, the src homology 3 (SH3) domain of insulin receptor substrate p53 (IRSp53) precipitated WAVE2 from platelet lysates more efficiently than did profilin I. The opposite was true for WAVE1, and neither precipitated WAVE3, suggesting that WAVE isoforms have different affinities to these ligands, while the SH3 domain of abl binds to all 3 isoforms. By mass spectrometry, we found that the proteins, which reportedly interact with WAVE/Scars, are present in platelets.
Thus, we have effectively employed mass spectrometry to demonstrate hitherto unreported proteins in platelets and their interactions.