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Regulation of human platelet Rac1 activation through interaction with calmodulin
Author(s) -
Bhullar Rajinder Pal,
Elsaraj Sherif M
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.306
Subject(s) - rac1 , calmodulin , platelet activation , thrombin , platelet , microbiology and biotechnology , motility , chemistry , biochemistry , cytoskeleton , biology , signal transduction , cell , immunology , enzyme
Rac1 belongs to the Rho family of small GTP‐binding proteins and has been implicated in lamellipodia formation. Calmodulin (CaM), a ubiquitous Ca 2+ ‐binding protein, has been shown to interact with and regulate the activity of several small GTP‐binding proteins. In the present study, we have investigated if CaM interacts with Rac1 and regulates its activation. Thus, Rac1 was expressed as a GST fusion protein (GST‐Rac1). In vitro pull down assay demonstrated that CaM from platelet lysate interacts in a Ca 2+ ‐dependent manner with GST‐Rac1. In the reverse experiment, CaM coupled to Sepharose beads was used to pull down Rac1 from platelet lysate in a Ca 2+ ‐dependent manner. Pure CaM was also able interact with Rac1 demonstrating that Rac1‐CaM interaction was direct. Using CaM database analysis a 14 amino acid sequence in Rac1 was identified as the CaM binding region. The scrambled form of the peptide did not bind CaM. The role of CaM in Rac1 activation was investigated using freshly isolated human platelets. Maximal activation of platelet Rac1 in response to thrombin occurred at ∼60 sec. Incubation with the CaM inhibitor, W7, prior to thrombin challenge, abolished thrombin induced activation of platelet Rac1. The results demonstrate that in platelets, binding of CaM to Rac1 increases its activation and that CaM may play an important role in the regulation of cytoskeleton remodelling. Supported by Heart & Stroke Fdn of Manitoba.