The adamalysins (ADAMs) are transmembrane glycoproteins involved in cell adhesion and proteolytic ectodomain processing of cytokines and adhesion molecules. Many ADAM cytoplasmic domains are prolinerich and have potential phosphorylation sites. We show here that the cytoplasmic domain of ADAM15, metargidin, can interact specifically with Src family proteintyrosine kinases (PTKs) and the adaptor protein Tyr 735 . These data demonstrate selective, phosphorylation-dependent interactions of ADAM15 with Src family PTKs and Grb2, which highlight the potential for integration of ADAM functions and cellular signaling.
We report the characterization of two members of a gene family from Arabidopsis that encode, respectively, cytosolic (cPMSR) and plastid-targeted (pPMSR) isoforms of the oxidative-stress-repair enzyme peptide methionine sulfoxide reductase. Overexpression of these proteins in Escherichia coli confirmed that each had PMSR enzyme activity with a synthetic substrate,N-acetyl-[3H]-methionine sulfoxide, or a biological substrate, α-1 proteinase inhibitor. The pPMSR was imported into intact chloroplasts in vitro with concomitant cleavage of its approximately 5-kD N-terminal signal peptide. The two PMSR isoforms exhibited divergent pH optima, tissue localization, and responses to developmental and environmental effects. Analysis of the Arabidopsis database indicated that there are probably at least twop-pmsr-like genes and three c-pmsr-like genes in the Arabidopsis genome. Expression of thep-pmsr genes and their protein products was restricted to photosynthetic tissues and was strongly induced following illumination of etiolated seedlings. In contrast, thec-pmsr genes were expressed at moderate levels in all tissues and were only weakly affected by light. Exposure to a variety of biotic and abiotic stresses showed relatively little effect onpmsr gene expression, with the exception of leaves subjected to a long-term exposure to the cauliflower mosaic virus. These leaves showed a strong induction of the c-pmsrgene after 2 to 3 weeks of chronic pathogen infection. These data suggest novel roles for PMSR in photosynthetic tissues and in pathogen defense responses in plants.
Adamalysins [a disintegrin and metalloproteinase (ADAM)] are a family of cell surface transmembrane proteins that have broad biological functions encompassing proteolysis, adhesion, and cell signal regulation. We previously showed that the cytoplasmic domain of ADAM-15 interacts with Src family protein tyrosine kinases and the adaptor protein growth factor receptor binding protein 2 (Grb2). In the present study, we have cloned and characterized four alternatively spliced forms of ADAM-15, which differ only in their cytoplasmic domains. We show that the four ADAM-15 variants were differentially expressed in human mammary carcinoma tissues compared with normal breast. The expression of the individual isoforms did not correlate with age, menopausal status, tumor size or grade, nodal status, Nottingham Prognostic Index, or steroid hormone receptor status. However, higher levels of two isoforms (ADAM-15A and ADAM-5B) were associated with poorer relapse-free survival in node-negative patients, whereas elevated ADAM-15C correlated with better relapse-free survival in node-positive, but not in node-negative, patients. The expression of ADAM-15A and ADAM-15B variants in MDA-MB-435 cells had differential effects on cell morphology, with adhesion, migration, and invasion enhanced by expression of ADAM-15A, whereas ADAM-15B led to reduced adhesion. Using glutathione S-transferase pull-down assays, we showed that the cytoplasmic domains of ADAM-15A, ADAM-15B, and ADAM-15C show equivalent abilities to interact with extracellular signal-regulated kinase and the adaptor molecules Grb2 and Tks5/Fish, but associate in an isoform-specific fashion with Nck and the Src and Brk tyrosine kinases. These data indicate that selective expression of ADAM-15 variants in breast cancers could play an important role in determining tumor aggressiveness by interplay with intracellular signaling pathways. (Mol Cancer Res 2008;6(3):383 -94)
SummaryThe oxidation of methionine residues in proteins to methionine sulfoxides occurs frequently and protein repair by reduction of the methionine sulfoxides is mediated by an enzyme, peptide methionine sulfoxide reductase (PMSR, EC 1.8.4.6), universally present in the genomes of all so far sequenced organisms. Recently, ®ve PMSR-like genes were identi®ed in Arabidopsis thaliana, including one plastidic isoform, chloroplast localised plastidial peptide methionine sulfoxide reductase (pPMSR) that was chloroplast-localized and highly expressed in actively photosynthesizing tissue (Sadanandom A et al., 2000). However, no endogenous substrate to the pPMSR was identi®ed. Here we report that a set of highly conserved methionine residues in Hsp21, a chloroplast-localized small heat shock protein, can become sulfoxidized and thereafter reduced back to methionines by this pPMSR. The pPMSR activity was evaluated using recombinantly expressed pPMSR and Hsp21 from Arabidopsis thaliana and a direct detection of methionine sulfoxides in Hsp21 by mass spectrometry. The pPMSR-catalyzed reduction of Hsp21 methionine sulfoxides occurred on a minute time-scale, was ultimately DTT-dependent and led to recovery of Hsp21 conformation and chaperone-like activity, both of which are lost upon methionine sulfoxidation (Ha È rndahl et al., 2001). These data indicate that one important function of pPMSR may be to prevent inactivation of Hsp21 by methionine sulfoxidation, since small heat shock proteins are crucial for cellular resistance to oxidative stress.
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