Phosphopeptide substrates

The co-crystal structure of yeast Cdc4 and its phosphorylated substrates, as well as that of yS-Trcp and its phosphopeptide substrate derived from j6-catenin, have... 

Figure 5 (a) Proposed catalytic mechanism of protein tyrosine phosphatases, (b) Structure of PTPl B (gray) with a phosphopeptide substrate (goid) showing the relative positions of catalytic residues to the phosphotyrosine moiety. 

Some properties of phosphopeptides make them preferable to the native phosphoprotein substrates for use with phosphate detection systems. The values of for peptide substrates are two to three orders of magnitude larger than for protein substrates and allow setting assays with an appropriate substrate concentration using standard phosphate detection approaches. In addition, short synthetic peptides are inexpensive and easy to obtain. Nonetheless, although phosphopeptide substrates are clearly useful in exploring interactions in the immediate vicinity of the phosphatase active site, they are unable to probe distant (allosteric) sites. 

Competition binding analysis of a variety of phosphopeptides derived from different Cdc4 substrates revealed a range of affinities. For example. Sic IP = 24 pM, FarlP = 2.7 pM, Gcn4 = 0.8 pM. Importantly, multi-site phosphorylation dependent interactions with Cdc4 can be recapitulated with synthetic concatamers of low affinity CPD sites based on either the regions T45 or S76 sites in Sicl. 

Dunkley PR, Robinson PJ (1986) Depolarization-dependent protein phosphorylation in synapto-somes mechanisms and significance. Prog Brain Res 69 273-93 Dunkley PR, Baker CM, Robinson PJ (1986) Depolarization-dependent protein phosphorylation in rat cortical synaptosomes characterization of active protein kinases by phosphopeptide analysis of substrates. J Neurochem 46 1692-1703... 

Antibodies coated onto MTP wells capture kinase or phosphatase substrate phosphorylation state is detected by anti-phosphopeptide antibody coupled to detector dye can be read by time-resolved fluorescence (DELFIA) technique... 

Kinase substrates can become resistant to the actions of proteases due to their phosphorylations. Thus, the fluorescence quench assays (described in Chapter 2 covering protease assays) can be used to measure kinase activity. The assays can be viewed as coupled because they require a second enzyme to convert a product or substrate into a detectable signal. With kinase assays, the formation of phosphopeptide inhibits the protease action on the peptide and the signal remains quenched and therefore decreased (Rodems et al., 2002). Inhibiting the kinase results in increases in protease sensitivity and in signal. 

Phosphotyrosine phosphatase activity was measured at 30°C in a total volume of 100 /x.L containing 24 mM imidazole (pH 7.2), 1 mM EDTA, 1 mM dithiothreitol, 100 jug of bovine serum albumin, 50 fiM dansyl phosphopeptide, and extracts containing enzyme activity. After 15 minutes, the reaction was terminated by the addition of 20 /x.L of 30% (w/v) trichloroacetic acid. The mixture was centrifuged before injection for HPLC analysis. The reaction was linear with respect to both time and enzyme concentration up to at least 30% substrate conversion. 

JH Till, RS Annan, SA Carr, WT Miller. Use of synthetic peptide libraries and phosphopeptide-selective mass spectrometry to probe protein kinase substrate specificity. J Biol Chem 269 7423-7428, 1994. 

Although mechanistically still elusive, an interaction of polycations both with the phosphoprotein substrate and the enzyme is required to facilitate PP2A-mediated dephosphorylation [125,126]. That may also explain why polycations are without any effect towards phosphopeptide PP2A substrates [127]. To date, it remains unclear whether the obvious effects of polycations on PP2A activity in vitro may become of future therapeutic value. 

The substrates of phosphoprotein phosphatases range from simple phosphopeptides to phosphorylated enzymes involved in many biological processes. Based on substrate specificity and sequence homology, these enzymes fall into two large families, the PTPs and the protein serine/threonine phosphatases (PPs). As their name implies, the PPs dephosphorylate pSer and phosphothreonine (pThr) residues. These enzymes have binuclear metal centers and catalyze a phosphoryl transfer from the substrate directiy to water. The PPs are distinguished from the PTPs, which do not contain metal ions and dephosphorylate phosphotyrosine (pTyr) residues through a phosphoenzyme intermediate. 

Olejniczak, E. T., Zhou, M. M., Fesik, S. W. 1997. Changes in the NMR-derived motional parameters of the insulin receptor substrate 1 phosphotyrosine binding domain upon binding to an interleukin 4 receptor phosphopeptide. Biochemistry 36,4118-4124. 

Protein phosphorylation mediated by protein kinases is the principal mechanism by which eukaryotic cellular processes are modulated by external physiological stimuli (1). Phosphopeptides are essential tools for the study of this process, serving as model substrates for phosphatases, as antigens for the production of antibodies against phosphorylated proteins, and as reference compounds for determining their physical parameters. The development of methods for the production of phosphopeptides has consequently attracted considerable interest over the last few years, and these endeavours have yielded reliable procedures which have now made their synthesis routine. 

Fig. 2. Schematic depicting the method to discover substrates for protein kinases. Beads with unique peptide sequences are incubated with a protein kinase and y[ P] ATP. After washing, the beads are spread out in agarose and autoradiographed. Radioactive beads are isolated, tested by quantitative methods, and the presumptive phosphopeptides are sequenced.

Using these methods, the structure of the PTB domain of the IRS-1 protein was found to be similar to phosphopeptide-binding regions of several other proteins. Once the structural details were known, the different binding specificities could be compared and rationalized based on the interactions with their substrates. 

---