Molecular interactions between ADAM12 and the non-receptor tyrosine kinase c-Src
Abstract:ADAM12 belongs to the family of ADAMs (A Disintegrin And Metalloproteases), which are multidomain proteins, involved in cell adhesion and ecto-domain shedding of a variety of cytokines and growth factors. Compelling evidence for an important role of ADAM12 in cancer is accumulating – i.e. ADAM12 is found markedly upregulated in several types of human tumors and the level of ADAM12 in urine from breast and bladder cancer patients correlates with disease stage, suggesting that ADAM12 is a potential prognostic biomarker. In agreement with these findings, we recently reported that ADAM12 increases tumor aggressiveness in a mouse model of breast cancer. Yet, the molecular mechanisms behind ADAM12 activities in cancer remain elusive.
Human ADAM12 exists in two splice variants; a long form, ADAM12-L as well as a secreted form, ADAM12-S. ADAM12-L encompasses pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domains, where ADAM12-S lacks the transmembrane and cytoplasmic parts. Little is known about the structure and function of the cytoplasmic domain, but interestingly, mouse ADAM12-L has been shown to bind the proto-oncoprotein and non-receptor tyrosine kinase c-Src, raising the question whether ADAM12 and c-Src activities in cancer are linked. In order to study this in more detail, a detailed structure-function analysis of this interaction has been initiated. Using GST-pull down and competitive peptide inhibition experiments, we identified two separate binding sites in the cytoplasmic tail of human ADAM12-L that interacts with the SH3-domain of c-Src. While both sites comprise a consensus class I proline-rich Src-SH3 binding motif, a pronounced difference in binding affinity was observed, possibly due to steric availability in the 3-dimentional folding. Co-expression of ADAM12-L with wildtype c-Src and a constitutively active mutant form of c-Src lead to ADAM12-L tyrosine phosphorylation, whereas no effect of a kinase-dead c-Src mutant was detected. Surprisingly, ADAM12-mediated ecto-domain shedding of epidermal growth factor (EGF) was independent of Src-kinase activity. Current studies aim at unraveling the functional outcome of the interaction between ADAM12-L and c-Src and a potential mutual role in cancer.Acknowledgements: Grant support from the Danish Cancer Society, the Danish Medical Research Council, Novo Nordisk, Friis and Haensch Foundations.
Document Type: Abstract
Affiliations: 1: Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia PA, USA 2: Department of Biomedical Sciences & Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen Biocenter, Ole MaaløesVej 5, 2200 Copenhagen N, Denmark
Publication date: May 1, 2008