Down-regulation of either Gab1 or Gab2 by siRNAs (small interfering RNAs) effectively inhibited the EGF-stimulated ERK activation pathway and cell migration. activation. It was found that EGF induced a similar amount of SHP2CGab1 and SHP2CGab2 complexes. Expression of either SHP2-binding defective Gab1 or Gab2 mutant blocked EGF-induced ERK activation. Down-regulation of either Gab1 or Gab2 by siRNAs (small interfering RNAs) effectively inhibited the Valifenalate EGF-stimulated ERK activation pathway and cell migration. Interestingly, the inhibitory effect of Gab1 siRNA could be rescued not only by expression of an exogenous mouse Gab1 but also by an exogenous human Gab2 and vice versa, but not by IRS1 (insulin receptor substrate 1). These results reveal that Gab2 plays a pivotal role in the EGF-induced ERK activation pathway and that it can complement the function of Gab1 in the EGF signalling pathway. Furthermore, Gab1 and Gab2 are critical signalling threshold proteins for ERK activation by EGF. growth factor receptor bound protein 2-associated protein 1; GenBank? accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”BC007483″,”term_id”:”13938652″BC007483) was amplified by PCR using pfuUltra high-fidelity DNA polymerase (Stratagene, La Jolla, CA, U.S.A.). The PCR fragment was subcloned into the pcDNA3.1-FLAG vector [5] between XhoI and XbaI sites to generate the expression plasmid for a FLAG-tagged mouse Gab1. The wild-type and SHP2-binding defective (Gab2F) mouse Gab2 have been described previously [14]. For preparation of an expression vector for human Gab2, we first prepared the N-terminal 136?bp coding region by nested PCR using human K562 leukaemia cDNA as the template. Nucleotides 112C2028 (the last coding nucleotide) of the human Gab2 coding sequence was amplified by PCR from the KIAA0571 plasmid (mRNA for KIAA0571 protein; GenBank? accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AB011143″,”term_id”:”3043665″AB011143) kindly provided by Dr T. Nagase (Kazusa DNA Research Institute, Kisarazu, Chiba, Japan). The two overlapping PCR fragments covering the complete human Gab2 coding region were annealed and used as the template for PCR Valifenalate to produce a complete Gab2 coding cDNA. The PCR fragment was digested with KpnI and XbaI and then subcloned into pcDNA3.1-FLAG. An expression vector for FLAG-tagged IRS1 was generated by PCR cloning of the human IRS1 coding sequence from “type”:”entrez-nucleotide”,”attrs”:”text”:”BC053895″,”term_id”:”31753080″BC053895 (from Open Biosystems, Huntsville, AL, U.S.A.) into pcDNA3.1-FLAG. The cloning sites were HindIII and XbaI. DNA sequences were verified by sequence analysis. PCR primer sequence information will Mouse monoclonal to CD48.COB48 reacts with blast-1, a 45 kDa GPI linked cell surface molecule. CD48 is expressed on peripheral blood lymphocytes, monocytes, or macrophages, but not on granulocytes and platelets nor on non-hematopoietic cells. CD48 binds to CD2 and plays a role as an accessory molecule in g/d T cell recognition and a/b T cell antigen recognition be provided upon request. Preparation of plasmid-based siRNAs The target sequence for human Gab2 is 1652-GGGCCAACCACACCTTCAACT (the number indicates the location of the first nucleotide of the target sequence in the human Gab2 coding region). Oligonucleotides 5-AACCACACCTTCAACTAAGCTTAGTTGAAGGTGTGGTTGGCCCTTTTTG and 5-AATTCAAAAAGGGCCAACCACACCTTCAACTAAGCTTAGTTGAAGGTGTGGTTGGCC containing the siRNA coding sequence and an AAGCTT loop were annealed and cloned between the ApaI and EcoRI sites of plasmid pBS/U6 [28] to generate pGab2siRNA-1652. To generate a plasmid (pGab2siRNA) containing two copies of the Gab2 siRNA transcription units, the siRNA transcription unit was excised from pGab2siRNA-1652 by BamHI digestion. The DNA fragment was made bluntended by treatment with the Klenow fragment of DNA polymerase, and inserted into the SmaI site of pGab2siRNA-1652. An siRNA plasmid targeting human Gab1 sequence Valifenalate 976-GGAACCTTGGGACAGACAT and containing an ACA loop was also constructed in pBS/U6. To facilitate the preparation of other siRNAs, we made a pBSU6-BbsI vector. Plasmid pBS/U6 [28] was digested with ApaI and the 3-protruding ends were converted into blunt-ends with the Klenow fragment of DNA polymerase I. The plasmid was then digested with EcoRI. Oligonucleotides 5-pTGGTCTTCGTCG (top) and 5-pAATTCGACGAAGACCA (bottom, BbsI recognition sequence underlined) were annealed and inserted between the blunt-end and the EcoRI site to make the pBSU6-BbsI vector. siRNA plasmids targeting human Gab1 siRNA sequences 874-GGGACATCGAGTGTAGAGACT and 1135-GGGATGTCGCCTTCACGTAGT were prepared in pBSU6-BbsI. The loop for both of these siRNAs was AAGCTT. A plasmid containing triple siRNA transcription units (pGab1siRNA) for Gab1-874, Gab1-1135 and Gab1-976 was constructed by sequential insertion of blunt-ended BamHI fragments containing the siRNA transcription units into the SmaI site of the plasmid containing siRNA for Gab1-874. A plasmid containing siRNA for both human Gab1 and Gab2 (pGab1/pGab2siRNA) was generated by subcloning the blunt-ended BamHI fragment containing pGab2siRNA-1652 into pGab1siRNA. Again, oligonucleotide sequence information for the construction of these siRNA vectors is available upon request. Preparation of adenovirus-based siRNA and adenovirus encoding Gab1 The AdEasy XL adenoviral vector system (Stratagene) was used to generate adenovirus-based siRNAs for human Gab1 and Gab2. The three Gab1 siRNA transcription units, two Gab2 siRNA transcription units, and the U6 promoter transcription unit were excised from pGab1siRNA, pGab2siRNA and pBS/U6 respectively by XbaI digestion. These DNA fragments were subcloned into the XbaI site of pShuttle. Recombinant adenoviral plasmids were isolated.