The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp6Orc and pp590 ', via ing sequence motif (43, 44). The specificity of such binding is dictated by the residues flanking the site of tyrosine phosphorylation as well as the structural characteristics of a particular SH2 domain (12,54). Examples drawn from growth factor receptor PTK signalling illustrate the importance of SH2-phosphotyrosine interactions in the transmission of cytoplasmic signals. For example, receptor PTK autophosphorylation can recruit SH2-containing substrates to the receptor complex, thereby facilitating their phosphorylation. In turn, the phosphorylation of certain substrates appears to be critical for the regulation of their activities (for example, the growth factordependent activation of phospholipase C&y [13,23,40]). SH2-phosphotyrosine interactions also provide a mechanism by which cytosolic enzymes whose substrates are present in cell membranes can be translocated to the proximity of their substrates (for example, phospholipase C&y and phosphatidylinositol 3-kinase, cytosolic enzymes whose substrates are membrane lipids [9,26,37], and Sos, a cytosolic protein that functions as a guanine nucleotide exchange factor for the membrane-associate protein p2lras [34]). Binding of a tyrosinephosphorylated protein to an SH2 domain-containing enzyme can lead directly to an increase in enzymatic activity. For example, binding of a tyrosine-phosphorylated peptide to the SH2 domain of the regulatory 85-kDa subunit of phosphatidylinositol 3-kinase (P13K) induces conformational changes in p85 and the concomitant increase in enzymatic activity of the holoenzyme (2,6,35,41). Finally, SH2-phosphotyrosine interactions may be critical for the negative regulation of enzymatic activity. For example, c-Src kinase activity is regulated by the binding of its SH2 domain to the C-terminal regulatory site of tyrosine phosphorylation, 8). Thus, phosphotyrosine-SH2 interactions not only mediate protein-protein complex formation but are also intimately involved in the regulation of the activity of a number of signalling molecules.The PTK pp125FAK is phosphorylated on tyrosine in response to the engagement of cell surface integrins with com-1680 on April 2, 2019 by guest
Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd‐I (positions 1–905) comprising the N‐terminal globule and rod‐like domain and Nd‐II corresponding mainly to the C‐terminal globule (position 906–1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N‐terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N‐terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31–56 kDa) connected by either a rod‐like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd‐I binding to collagen IV and fragment Nd‐II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd‐I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.
As more data are generated from proteome and transcriptome analyses of snake venoms, we are gaining an appreciation of the complexity of the venoms and, to some degree, the various sources of such complexity. However, our knowledge is still far from complete. The translation of genetic information from the snake genome to the transcriptome and ultimately the proteome is only beginning to be appreciated, and will require significantly more investigation of the snake venom genomic structure prior to a complete understanding of the genesis of venom composition. Venom complexity, however, is derived not only from the venom genomic structure but also from transcriptome generation and translation and, perhaps most importantly, post‐translation modification of the nascent venom proteome. In this review, we examine the snake venom metalloproteinases, some of the predominant components in viperid venoms, with regard to possible synthesis and post‐translational mechanisms that contribute to venom complexity. The aim of this review is to highlight the state of our knowledge on snake venom metalloproteinase post‐translational processing and to suggest testable hypotheses regarding the cellular mechanisms associated with snake venom metalloproteinase complexity in venoms.
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