The second DDR binding partner identified here Shp
The second DDR1-binding partner identified here, Shp-2, which was not found in the two-hybrid screen but using a bioinformatics approach, is also a mediator of cell migration. Since Shp-2 has two SH2 domains, as well as phosphotyrosine phosphatase enzymatic activity and two C-terminal tyrosines targeted by upstream kinases, its mode of regulation is even more intricate , , . Mutations in the gene coding for Shp-2 were found in a number of human cancers, including lung and colon tumors, as well as leukemias, neuroblastomas and the genetic disorder Noonan-syndrome . Depending on the cellular context, binding of Shp-2 to activated growth factor receptors can lead to dephosphorylation of a Ras-GAP binding site, thereby attenuating Ras-GTP turnover and enhancing Ras-mediated cell migration and proliferation . However, Shp-2-induced dephosphorylation of other signaling intermediates, such as Stat5, a downstream mediator of the interleukin, growth factor and prolactin receptors, can terminate a signaling event . Interestingly, in a separate study we found that prolactin-induced Stat5 phosphorylation and nuclear translocation is severely diminished in DDR1-null mammary gland ergosterol and tissues, while overexpression of DDR1 enhanced Stat5 activity . Therefore, our findings suggest that Shp-2 downstream of DDR1 has a dual function in the mammary gland: it triggers cell differentiation and migration during puberty and pregnancy while sustaining DDR1-mediated signaling during lactation.
We show here that Shp-2 binds to phosphotyrosine-740 in the cytoplasmic domain of DDR1 in a collagen-dependent manner (Figs. 3A and 4A). The peptide sequence ISpYPML neighboring tyrosine 740 matches well with the immuno-receptor tyrosine-based inhibitory motif (ITIM)-consensus sequence [I/V/L]XpYXX[I/V/L] found in large a number of proteins, many of them belonging to the immune receptor inhibitor and cell adhesion receptor family . Importantly, Shp-2 was found to associate with a diverse range of signal-regulatory receptors, including PECAM-1, FcgammaRIIb, SRPSs and gp49B , , . It is tempting to speculate that recruitment of active Shp-2 into the DDR1 signaling complex also results in dephosphorylation of molecules within the complex, thereby generating an inhibitory signal that limits cell proliferation or differentiation. Evidence from DDR1-null mice showing hyperproliferative mammary gland tissue and enhanced growth of mesangial cells in the absence of DDR1 is in support of this hypothesis , .
Our finding of enzymatic-inactive Shp-2 being phosphorylated upon collagen stimulation suggests that DDR1 recognize one or both of the C-terminal phosphorylation sites in Shp-2, which were found to serve as docking sites for the Grb2 and Gab2 adaptor molecules , , . Binding of Shp-2 to DDR1 most likely enhances the enzymatic activity of the phosphatase, thereby directly triggering downstream events. In future work we will address this by overexpressing the Shp-2 C463A mutant in normal and transformed breast epithelial cells as well as by re-expressing the DDR1-Y740F mutant in knockout cells.
Introduction Basement membranes are important barriers for filtration of macromolecules. This function is particularly apparent in the glomerular basement membrane (GBM). The GBM is a fusion product of two basement membranes built by endothelial and epithelial cells (podocytes) (Abrahamson et al., 2009). Fenestrations within the endothelial cell layer allow direct access of plasma proteins to the GBM (Kriz 2008). The intricate network of foot processes reaching out from the podocytes further contributes to the filtration unit. The podocytes' foot processes are linked by a slit-diaphragm, originally described as a zipper like structure by Rodewald and Karnovsky (1974). Since this early work much has been learned about the molecules that generate its unique permselectivity and mechanical stability of the filtration unit within the kidney (Kriz 2008). One major constituent of the GBM is the network of type IV collagen, large molecules with interrupted triple-helical stretches, aggregating into a highly ordered network via their N-terminal and C-terminal globular domains (Hudson et al., 2003). Type IV collagen plays a part in a variety of protein–protein interactions, including laminins, proteoglycans, nidogens, as well as in cell attachment via α1β1 integrin (Cosgrove et al., 2000). In mammals including humans and mice, six genetically distinct type IV collagen α-chains α1–α6 have been identified, encoded by six separate genes COL4A1–COL4A6 (Hudson et al., 2003). While the immature GBM consists of α1/α1/α2(IV) chains, α3/α4/α5(IV)-heterotrimers are found in the mature GBM (Miner and Sanes, 1994, Borza et al., 2002, Hudson et al., 2003). Mature GBM contains numerous disulfide-bonds providing advanced mechanical strength needed for filtration of about 150l of primary urine per day.