TY - JOUR
T1 - Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening
AU - Wu, Chenggang
AU - Ma, Mike Haiting
AU - Brown, Kevin R.
AU - Geisler, Matt
AU - Li, Lei
AU - Tzeng, Eve
AU - Jia, Christina Y.H.
AU - Jurisica, Igor
AU - Li, Shawn S.C.
PY - 2007/6
Y1 - 2007/6
N2 - Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCγ1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCγ1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCγ1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.
AB - Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCγ1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCγ1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCγ1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.
KW - HPK1
KW - Interactome
KW - Peptide array
KW - PLCγ1
KW - SH3 domain
UR - http://www.scopus.com/inward/record.url?scp=34250379613&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34250379613&partnerID=8YFLogxK
U2 - 10.1002/pmic.200601006
DO - 10.1002/pmic.200601006
M3 - Article
C2 - 17474147
AN - SCOPUS:34250379613
SN - 1615-9853
VL - 7
SP - 1775
EP - 1785
JO - Proteomics
JF - Proteomics
IS - 11
ER -