Protein kinase Substrates

The next class of cosubstrates for kinases consists of proteins. Protein kinases are very important in the regulation of cellular processes. Investigations of both these mechanisms and the roles that the protein kinases play might be aided by the use of selective inhibitors of these kinases. One indication of just how little is known about the characteristics of protein kinase-substrate interactions is the fact that many of these enzymes are named by their activators rather than by their substrates. 

Evans, J. P, Wickremasmghe, R. G, and Hoffbrand, A. V. (1987) Detection of tyrosine protein kinase substrates in fresh leukemia cells and normal blood cells using an immunoblotting technique. Leukemia 1, 782—785... 

Radionuclide-based screening for protein kinase substrates is a quick and effective method for determining peptide substrates for protein kinases.28-31 The method presented here is based on the screening of random synthetic combinatorial peptide libraries with a protein kinase and is divided into two main parts (1) the phosphorylation of the peptide bead library and (2) the recovery and characterization of positive beads. 

Parang K, Kohn JA, Saldanha SA, Cole PA. Development of photo-crosslinking reagents for protein kinase-substrate interactions. FEBS Lett 2002 520 156-160. 

Tegge, W., Frank, R., Hofmann, F., and Dostmann, R.G. (1995) Determination of cyclic nucleotide-dependent protein kinase substrate specificity by the use of peptide libraries on cellulose paper. Biochemistry 34, 10569-10577. 

Identification of cAMP- and cCMP-Dependent Protein Kinase Substrates in Human Platelets... 

Table 11.1 Identification and function of cAMP- and cGMP-dependent protein kinase substrates in human platelets.

B-Cell epitope mapping (foot-and-mouth disease, TMV antigen, shrimp allergen) T-Cell epitope mapping (type 1 diabetes, alloreactive T-cell) protein kinase substrate/inhibitor (protein kinase I) MHC class I molecule binding peptides and epitope mapping for monoclonal and polyclonal antibodies. 

Protein kinase substrate/inhibitor (Csk, 3 bp2, Fps Fes, Grb-2, Hep, She, Syk, Vav, Zap-70) Fpitope mapping for monoclonal antibodies SH2 domain-binding peptides and kinase domains of protein tyrosine kinases... 

BUSIELO XR, LODBETTBt JA, BaRBACID M. Product of vav proto-oncogene defines a new dass of tyrosine protein kinase substrates. Nature3S6 68-71,1992. 

Fisher TH, Gatling MN, LaCAL JC, White GC. Rap IB, a cAMP-dependent protein kinase substrate, assodates with the platdetcytoslceleton.yPio/C%em 265 19405-19408,19. ... 

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. 

Many protein kinases are maintained in an inhibited state by an autoinhibitory domain within the protein or as a separate inhibitory subunit (Kemp et al., 1994). Activation thus involves exposure of the catalytic site so that the protein substrate may bind and be phosphorylated. A number of autoinhibitory sequences have structural similarities to the consensus phosphorylation site in the respective protein kinase substrates. Thus, the enzyme s active site may be ster-ically blocked by the autoinhibitory domain via a pseudosubstrate mechanism, where residues in the catalytic core bind to residues in the autoinhibitory region or substrate. 

In this method, phosphotransferase activity is used to measure MAPK activity in extracts of smooth muscle tissue. MAPK-specific activity is detected using a synthetic peptide as a substrate in the phosphotransferase reaction. The use of myelin basic protein will result in the detection of all kinases that phos-phorylate this substrate (including MAPK, protein kinase C, and other protein kinases whereas the use of a synthetic peptide, designed as a proline-directed protein kinase substrate, results in the detection of MAPK-specific activity, exclusively (Adam et al., 1995 Clark-Lewis et ah, 1991). 

Peptide Pepscan systems www.pepscan.nl Arrays printed with 1200 protein kinase substrate peptides. 

Following the binding of protein kinase substrates by calmodulin... 

Chi NW, Lodish HF. Tankyiase is a golgi-associated min en-activated protein kinase substrate that interacts with IRAP in GLUT4 vesicles. J Biol Chem 2000 273(49) 38437-44. 

Fig. 5 Substrate specifity of the purified protein kinase substrate additions (a) purified chromatophores (30/Xg), (b) isolated B875 complexes (30/Xg), (c) histone V-S (15/ig).

INVESTIGATION OF THYLAKOID PROTEIN KINASE SUBSTRATE SPECIFICITY USING SYNTHETIC PEPTIDES. 

In this communication we present data demonstrating the relationship between synthetic peptide charge and efficiency as thylakoid protein kinase substrates. 

Investigation of Thylakoid Protein Kinase Substrate Specificity Using Synthetic Peptides 767... 

The low response to the addition of poly(l) poly(c) to the S-20 fraction of interferon-treated, mengovirus-infected cells, suggested that the location (or relative concentration) of some of the components of the phosphorylating system (protein kinase, substrate P67, phosphatase) was modified by the infection. In interferon-treated non-infected cells extracts, P67 was found in S-20 as well as in S-100. 

---