Protein interaction analysis of ST14 domains and their point and deletion mutants.
ST14 (suppression of tumorigenicity 14) is a transmembrane serine protease that contains a serine protease catalytic (SP) domain, an SEA domain, two complement subcomponent C1r/s (CUB) domains, and four low density lipoprotein receptor class A domains. Glutathione S-transferase fusion proteins with SP, CUB, and low density lipoprotein receptor domains and their corresponding mutants were generated to analyze protein interactions with these domains. Modified glutathione S-transferase pull-down assays demonstrated the interaction between the SP domain and hepatocyte growth factor activator inhibitor-1. With the same method, a CUB domain-interacting protein was isolated and turned out to be the transmembrane protein with epidermal growth factor-like and two follistatin-like domains 1 (TMEFF1). Quantitative real time PCR revealed that the expression of the TMEFF1 gene was dependent on the transfection of the ST14 gene in the RKO cell line. Our results also suggested that ST14 and TMEFF1 were co-expressed in the human breast cancer cell line MCF7, human placenta, kidney, and liver tissues. Interestingly, these two genes were co-up-regulated in kidney tumors versus normal tissues, consistent with our results that showed the dependence of TMEFF1 expression on ST14 in RKO cells. Finally, homology modeling studies suggested that TMEFF1 might form a complex with ST14 by an interaction between epidermal growth factor and CUB domains.
Ge
Weiting
W
College of Life Science, Zhejiang University, Hangzhou 310029, China.
Hu
Hanguang
H
Ding
Kefeng
K
Sun
Lifeng
L
Zheng
Shu
S
D000911:Antibodies, Monoclonal / Q000737:chemistry; D020134:Catalytic Domain; D002460:Cell Line; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000737:chemistry; D017353:Gene Deletion; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010920:Placenta / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D010455:Peptides; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D012697:Serine Endopeptidases; C485596:ST14 protein, human