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Cat # P0507
cDNA Clone
15875 Gaither Drive
pEGFP-PH Domain from
Btk
Gaithersburg, MD 20877
FAX. 301-560-4919
TEL. 301-330-5966
Email: [email protected]
Web: www.signagenlabs.com
This product is for laboratory research ONLY and not for diagnostic use
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.
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
 2006 SignaGen Laboratories
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
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Product shipped at ambient temperature.