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Custom cDNA Cloning Service
pEGFP-PH (Btk)
Catalog # P0507
Map and Sequence
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Introduction
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 4,5bisphosphate (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.
The PH domain of Bruton’s tyrosine kinase (Btk), which is mutated in the human disease Xlinked 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 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
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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.
Cloning Protocol:
1. PCR Amplification: The PH domain of Bruton’s tyrosine kinase (amino acids 1–177) were
amplified with the Advantage Klentaq polymerase mixture (CLONTECH) from human cDNAs
(Marathon cDNA from brain and K562 leukemia cells, CLONTECH) with the following primer
pair:
5’-CCAAGTCCTGGCATCTCAATGCATCTG-3’
5’-TGGAGACTGGTGCTGCTGCTGGCTC-3’
2. Cloning: The amplified product was subcloned into the pGEM-Easy T/A cloning vector
(Promega) and sequenced with dideoxy sequencing (Thermosequenase, Amersham Pharmacia
Biotech). A second amplification reaction was performed from these plasmids with nested
primers that contained restriction sites for appropriate cloning into the pEGFP-N1 plasmid
(CLONTECH) to preserve the reading frame. Plasmids were transfected into COS-7 or NIH 3T3
cells and analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blotting for
the presence of the GFP fusion constructs using a polyclonal antibody against GFP
(CLONTECH).
MAP and Restriction Analysis:
The cDNA of Btk-PH domain was inserted into pEGFP-N1 vector on EcoRI/BamHI. The insert
sequence is as follows with underlined protein coding region which translates Btk Ph domain
with 177 amino acids:
TCCAGAAAGAAGAAGCT ATG GCC GCA GTG ATT CTG GAG AGC ATC TTT CTG AAG CGA TCC CAA CAG
AAA AAG AAA ACA TCA CCT CTA AAC TTC AAG AAG CGC CTG TTT CTC TTG ACC GTG CAC AAA CTC
TCC TAC TAT GAG TAT GAC TTT GAA CGT GGG AGA AGA GGC AGT AAG AAG GGT TCA ATA GAT
GTT GAG AAG ATC ACT TGT GTT GAA ACA GTG GTT CCT GAA AAA AAT CCT CCT CCA GAA AGA CAG
ATT CCG AGA AGA GGT GAA GAG TCC AGT GAA ATG GAG CAA ATT TCA ATC ATT GAA AGG TTC
CCT TAT CCC TTC CAG GTT GTA TAT GAT GAA GGG CCT CTC TAC GTC TTC TCC CCA ACT GAA GAA
CTA AGG AAG CGG TGG ATT CAC CAG CTC AAA AAC GTA ATC CGG TAC AAC AGT GAC TTG GTT
CAG AAA TAT CAC CCT TGC TTC TGG ATC GAT GGG CAG TAT CTC TGC TGC TCT CAG ACA GCC AAA
AAT GCT ATG GGC TGC CAA ATT TTG GAG AAC AGG AAT GGA AGC TTA AAA CCG
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The construct was fully sequenced and the full length is usually sent in text file by Email. The
construct map is as follows:
The map of backbone of pEGFP-N1:
(EcoRI)GA ATTC CAGAAAGAAGAAGCTATG GCC GCA GTG ATT CTG GAG AGC ATC TTT CTG AAG CGA TCC
CAA CAG AAA AAG AAA ACA TCA CCT CTA AAC TTC AAG AAG CGC CTG TTT CTC TTG ACC GTG CAC AAA
CTC TCC TAC TAT GAG TAT GAC TTT GAA CGT GGG AGA AGA GGC AGT AAG AAG GGT TCA ATA GAT GTT
GAG AAG ATC ACT TGT GTT GAA ACA GTG GTT CCT GAA AAA AAT CCT CCT CCA GAA AGA CAG ATT CCG
AGA AGA GGT GAA GAG TCC AGT GAA ATG GAG CAA ATT TCA ATC ATT GAA AGG TTC CCT TAT CCC TTC
CAG GTT GTA TAT GAT GAA GGG CCT CTC TAC GTC TTC TCC CCA ACT GAA GAA CTA AGG AAG CGG TGG
ATT CAC CAG CTC AAA AAC GTA ATC CGG TAC AAC AGT GAC TTG GTT CAG AAA TAT CAC CCT TGC TTC
TGG ATC GAT GGG CAG TAT CTC TGC TGC TCT CAG ACA GCC AAA AAT GCT ATG GGC TGC CAA ATT TTG
GAG AAC AGG AAT GGA AGC TTA AAA CCG GAT CC (BamHI)
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Full Map of pEGF-PH (Btk)
TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAA
ATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATA
GGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC
AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTC
CTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA
TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACG
GGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAA
GCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGCGCTACCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCCAGAAA
GAAGAAGCTATGGCCGCAGTGATTCTGGAGAGCATCTTTCTGAAGCGATCCCAACAGAAAAAGAAAACATCACCTCTAAA
CTTCAAGAAGCGCCTGTTTCTCTTGACCGTGCACAAACTCTCCTACTATGAGTATGACTTTGAACGTGGGAGAAGAGGCA
GTAAGAAGGGTTCAATAGATGTTGAGAAGATCACTTGTGTTGAAACAGTGGTTCCTGAAAAAAATCCTCCTCCAGAAAGA
CAGATTCCGAGAAGAGGTGAAGAGTCCAGTGAAATGGAGCAAATTTCAATCATTGAAAGGTTCCCTTATCCCTTCCAGGT
TGTATATGATGAAGGGCCTCTCTACGTCTTCTCCCCAACTGAAGAACTAAGGAAGCGGTGGATTCACCAGCTCAAAAACG
TAATCCGGTACAACAGTGACTTGGTTCAGAAATATCACCCTTGCTTCTGGATCGATGGGCAGTATCTCTGCTGCTCTCAG
ACAGCCAAAAATGCTATGGGCTGCCAAATTTTGGAGAACAGGAATGGAAGCTTAAAACCGGATCCACCGGTCGCCACCAT
GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGT
TCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTG
CCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCA
GCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACA
AGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGAC
GGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGG
CATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCC
CCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAG
AAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAG
CGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCC
CCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCA
ATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCT
TAAGGCGTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAAT
AGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAG
AGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACC
ATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAG
CTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGT
GTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCG
GGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCT
GATAAATGCTTCAATAATATTGAAAAAGGAAGAGTCCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGT
GTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAG
TCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCC
GCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGG
CCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAGATCGA
TCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGA
GGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGC
CCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGC
CACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGC
CGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCAT
ACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGG
TCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCA
TGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCT
GGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGA
GCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATC
GCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAG
ATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAG
CGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCTAGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGG
AACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGT
CCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCC
CACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCCTCAGGTTACTC
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ATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGA
CCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCT
TTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCT
ACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAG
GCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGC
GATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTC
GTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGC
TTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGG
GGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGG
GGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT
TCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCATGCAT
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