1. Cat # P0507 Store at -20 0 C cDNA Clone pEGFP-PH Domain from Btk 15875 Gaither Drive Gaithersburg, MD 20877 FAX. 301-560-4919 TEL. 301-330-5966 Email: info@signagenlabs.cominfo@signagenlabs.com Web: www.signagenlabs.comwww.signagenlabs.com Description: Pleckstrin homology (PH) domains are about 120 amino acid-long protein modules that were first described in pleckstrin, the major protein kinase C substrate in platelets. PH domains have since been identified in several key regulatory proteins with characteristic structural features that include two orthogonal beta sheets that form alpha sandwich with an a helix at the COOH terminus, and variable loops that create a highly charged surface. It has been generally accepted that PH domains provide a structural basis for the interaction of certain regulatory proteins with membranes. Some PH domains bind with high affinity (low µM or nM K d ) to specific phosphoinositides such as phosphatidylinositol- 4,5-bisphosphate, PI-3,4-P2 or PI-3,4,5-P3. Binding to phosphoinositides may allow PH proteins to respond to lipid messengers for example by relocation to membranes. The C-termini of some PH domains have also been reported to bind the beta/gamma subunits of heterotrimeric G-proteins. For example, the PH domain of phospholipase C (PLC) delta binds inositol 1,4,5-tris-phosphate (Ins[1,4,5]-P3 ) and associates with lipid vesicles containing phosphatidylinositol 4,5-bisphosphate (PtdIns-[4,5] P2 ), but only weakly binds other inositol phosphates and PtdIns(4)P. Similarly, the PH domain of the Akt protein kinase appears to bind PtdIns(3,4)-P2 but not Ptd-Ins(4,5)-P2. Because different PH domains specifically bind specific phosphoinositides, PH domains (usually with a fluorescent tag like GFP) have widely used to label the metabolism and distribution of these phosphoinositides. cDNA of PH Domain (Btk) Tagged with GFP: The PH domain of Bruton’s tyrosine kinase (Btk), which is mutated in the human disease X-linked agammaglobulinemia, has been shown to interact with PI(3,4,5)P3 in vitro. It was found that the localization of expressed BtkPH-GFP in quiescent NIH 3T3 cells was indistinguishable from that of GFP alone, both being cytosolic as assessed by confocal microscopy. In NIH 3T3 cells coexpressing BtkPH-GFP and the epidermal growth factor receptor, activation of epidermal growth factor or endogenous platelet-derived growth factor receptors caused a rapid (<3 min) translocation of the cytosolic fluorescence to ruffle-like membrane structures. This response was not observed in cells expressing GFP only and was completely inhibited by treatment with the PI3-kinase inhibitors wortmannin and LY292004. Membrane-targeted PI3-kinase also caused membrane localization of BtkPH-GFP that was slowly reversed by wortmannin. When the R28C mutation of the Btk PH domain, which causes X-linked agammaglob-ulinemia, was introduced into the fluorescent construct, no translocation was observed after stimulation. In contrast, the E41K mutation, which This product is for laboratory research ONLY and not for diagnostic use  2006 SignaGen Laboratories confers transforming activity to native Btk, caused significant membrane localization of BtkPH-GFP with characteristics indicating its possible binding to PI(4,5)P2. This mutant, but not wild-type BtkPH-GFP, interfered with agonist-induced PI(4,5)P2 hydrolysis in COS-7 cells. These results show in intact cells that the PH domain of Btk binds selectively to 3-phosphorylated lipids after activation of PI3-kinase enzymes and that losing such binding ability or specificity results in gross abnormalities in the function of the enzyme. Therefore, the interaction with PI(3,4,5)-P3 is likely to be an important determinant of the physiological regulation of Btk and can be utilized to visualize the dynamics and spatiotemporal organization of changes in this phospholipid in living cells. Applications: Utilized for Btk activation and labeling of PI- (3,4,5)-P3 distribution and level in living cells. Especially suitable for real time visualization of Btk activation by confocal microscopy Storage: Upon arrival store this product at -20 0 C. Product shipped at ambient temperature.