The use of soybean trypsin inhibitors as phosphorylatable substrates for a rat liver protein kinase.

Phosphorylation by a cAMP-independent rat liver protein kinase of protein substrates containing the structural feature required by mammary gland casein kinase (-Ser-X-Glu/Asp) has been demonstrated. In particular, the Bowman-Birk Soybean trypsin inhibitor, which is characterized, like other legume protease inhibitors, by clusters of acidic residues near the C-terminal side of seryl residue(s), proved to be a good model substrate for the protein kinase. Its phosphorylation, involving the Ser 65 residue, is apparently hindered by the binding of trypsin, while it is stimulated by unfolding induced by reduction and subsequent carboxy-methylation.     

Synthetic beta-turn peptides as substrates for a tyrosine protein kinase

An attempt has been made at defining the secondary structural requirement for phosphorylation of substrates of a protein tyrosine kinase from the leukemia virus-transformed LSTRA cell line. An examination of the sites of phosphorylation of substrates of protein tyrosine kinases indicated a relatively high probability of the beta-turn as the secondary structural feature at these sites. We have, therefore, synthesized three tyrosine peptides: Ala-Pro-Tyr-Gly-NHCH3, Leu-Pro-Tyr-Ala-NHCH3, and Pro-Gly-Ala-Tyr-NH2, of which the first two peptides, but not the third, would be expected to contain the tyrosine residue in a beta-turn. Circular dichroism and infrared spectral data on the peptides confirmed this expectation. Phosphorylation data on the peptides by the tyrosine kinase showed that the two beta-turn peptides were phosphorylated with Vmax and Km values comparable to those of the 13-residue-long arginine-containing synthetic peptide substrate having a sequence homologous to the autophosphorylation site of the LSTRA kinase. The peptides used here contain the shortest sequence length among the reported synthetic peptide substrates for protein tyrosine kinases. Their preference for the beta-turn indicated that this conformation may serve as the recognition site for tyrosine phosphorylation.     

Aza-β-amino acid containing peptidomimetics as cAMP-dependent protein kinase substrates

Peptidomimetic analogs of the peptide RRASVA, known as the “minimal substrate” of the catalytic subunit of the cAMP-dependent protein kinase (PKA), were synthesized by consecutive replacement of natural amino acids by their aza-β³ analogs. The peptidomimetics were tested as PKA substrates and the kinetic parameters of the phosphorylation reaction were determined. It was found that the interaction of these peptidomimetics with the enzyme active center was sensitive to the location of the backbone modification, while the maximal rate of the reaction was practically not affected by the structure of substrates. The pattern of molecular recognition of peptidomimetics was in agreement with the results of structure modeling and also with the results of computational docking study of peptide and peptidomimetic substrates with the active center of PKA. It was concluded that the specificity determining factors which govern substrate recognition by the enzyme should be grouped along the phosphorylatable substrate, and such clustering might open new perspectives for pharmacophore design of peptides and peptide-like ligands.     

Nuclear substrates of protein kinase C.

Starting from the finding that, for neuronal cells, the nuclear-membrane-associated protein kinase C (PKC) is the so-called 'membrane inserted', constitutively active form, we attempted to identify substrates of this nuclear PKC. For this purpose, nuclear membranes and other subcellular fractions were prepared from bovine brain, and in-vitro phosphorylation was performed. Several nuclear membrane proteins were found, the phosphorylation of which was inhibited by specific PKC inhibitors and effectively catalyzed by added PKC. Combining the methods of two-dimensional gel electrophoresis, in-situ digestion, reverse-phase HPLC and microsequencing, two of these nuclear PKC substrates were identified; the known PKC substrate Lamin B2, which serves as a control of the approach and the nucleolar protein B23. Our data suggest, that, for B23, Ser225 is a site of phosphorylation by PKC.     

Characterization of endogenous substrates for novel-type protein kinase C as well as conventional-type protein kinase C in primary cultured mouse epidermal cells

In primary cultured mouse epidermal cells, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), induced changes in the phosphorylation levels of 10 proteins, termed KP-1 to 10, in two-dimensional PAGE. Seven of these proteins were phosphorylated and three were dephosphorylated. Similar changes were induced by other PKC activators, but not by inactive phorbol ester. Among these substrate proteins, phosphorylation of three proteins, i.e. KP-1 (pI 4.7/23,000 M_r), KP-2 (pI 4.7/20,700 M_r) and KP-10 (pI 4.7/25,000 M_r was markedly enhanced by PMA and inhibited by a potent PKC inhibitor staurosporine. In vitro phosphorylation studies and phosphoamino acid analysis, using these proteins as substrate and PKC preparations obtained from epidermal cell lysate, revealed that KP-1 and -2 were directly phosphorylated by Ca²⁺-, phospholipid-dependent protein kinase (conventional-type PKC; cPKC), but not by Ca²⁺-independent, phospholipid-dependent protein kinase (novel-type PKC; nPKC). On the other hand, KP-10 was mainly phosphorylated by nPKC in intact epidermal cells. These results indicate that cPKC and nPKC in epidermal cells have different substrate specificity for endogenous proteins and may induce different signal transduction.     

Distinct structural requirements of Ca2+/phospholipid-dependent protein kinase (protein kinase C) and cAMP-dependent protein kinase as evidenced by synthetic peptide substrates

Protein kinase C, purified to near homogeneity from the brain, has been tested toward a variety of synthetic peptide substrates including different phosphorylatable residues. While it proved totally inactive toward the tyrosyl peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly, as well as toward several more or less acidic seryl peptides, it phosphorylates with a Ca2+/phospholipid-dependent mechanism, at seryl and/or threonyl residues, many basic peptides, some of which are also good substrates for cAMP-dependent protein kinase (A-kinase). Among the peptides tested, however, the best substrate for protein kinase C, with kinetic constants comparable to those of histones, is the nonapeptide Gly-Ser-Arg6-Tyr, which is not a substrate for A-kinase. Moreover, although the peptide Pro-Arg5-Ser-Ser-Arg-Pro-Val-Arg is a good substrate for both kinases, its derivative with ornitines replacing arginines is phosphorylated only by protein kinase C. Some typical substrates of A-kinase on the other hand, like the peptides Phe-Arg2-Leu-Ser-Ile-Ser-Thr-Glu-Ser and Arg2-Ala-Ser-Val-Ala, are phosphorylated by protein kinase C rather slowly and with unfavourable kinetic constants. It is concluded that, while both protein kinase C and A-kinase need basic groups close to the phosphorylatable residues, their primary structure determinants are quite distinct.     

The use of N-methylated peptides and depsipeptides to probe the binding of heptapeptide substrates to cAMP-dependent protein kinase

Peptide 1, Leu-Arg-Arg-Ala-Ser-Leu-Gly, is an excellent substrate for cAMP-dependent protein kinase. While the importance of both arginines for effective enzyme-substrate interactions has been shown, it has not been known whether the kinase will catalyze phosphorylation of substrates which contain other than peptide bonds. We report that analogs of peptide 1 which contain depsi linkages replacing selected amide bonds are good protein kinase substrates. Therefore, with the possible exception of the serine amide proton, no peptide 1 amide hydrogens are involved in peptide-peptide or peptide-enzyme hydrogen bonding crucial to defining the high substrate activity of this peptide. It is thus unlikely that peptide 1 is bound by the protein kinase while in an alpha-helical or a beta-turn structure. Three peptides were found to be very poor substrates for protein kinase, those containing N-methyl amino acids in place of Ser5 or Leu6 and a peptide containing Pro in place of Leu6. These peptides are poor substrates for the enzyme possibly because they are unable to adopt a conformation necessary for catalysis of phosphoryl group transfer to occur or due to steric effects in the enzymatic active site.     

Identification of protein kinase substrates by proteomic approaches

This review describes the current status of proteomic approaches to identify kinase substrates, which may lead to valuable medical applications. It guides the reader towards various methods using 2DE and liquid chromatography-tandem mass spectrometry. Dynamic changes of phosphorylation during extracellular stimuli can be quantitatively monitored by both technologies. Among appropriate prefractionation procedures, the purification of phosphoproteins and phosphopeptides is an absolute step for success. The temporal change and stoichiometry of phosphorylation are the important criteria to evaluate the physiological meaning of the reaction. Kinase substrates can also be identified by in vitro phosphorylation systems employing protein arrays, fractionated lysates, genetically engineered kinases and phage libraries. The final section contains an expert opinion on the current strategies and the issues we are going to challenge in the next 5 years.     

Identification of protein kinase substrates by proteomic approaches

This review describes the current status of proteomic approaches to identify kinase substrates, which may lead to valuable medical applications. It guides the reader towards various methods using 2DE and liquid chromatography-tandem mass spectrometry. Dynamic changes of phosphorylation during extracellular stimuli can be quantitatively monitored by both technologies. Among appropriate prefractionation procedures, the purification of phosphoproteins and phosphopeptides is an absolute step for success. The temporal change and stoichiometry of phosphorylation are the important criteria to evaluate the physiological meaning of the reaction. Kinase substrates can also be identified by in vitro phosphorylation systems employing protein arrays, fractionated lysates, genetically engineered kinases and phage libraries. The final section contains an expert opinion on the current strategies and the issues we are going to challenge in the next 5 years.     

Characterization of bovine aortic protein kinase C with histone and platelet protein P47 as substrates.

A Ca2+- and phospholipid-dependent protein kinase (protein kinase C) was partially purified from the media of bovine aortas by chromatography on DEAE-Sephacel and phenyl-Sepharose. Enzyme activity was characterized with both histone and a 47 kDa platelet protein (P47) as substrates, because the properties of protein kinase C can be modified by the choice of substrate. Both phosphatidylserine and Ca2+ were required for kinase activity. With P47 as substrate, protein kinase C had a Ka for Ca2+ of 5 microM. Addition of diolein to the enzyme assay caused a marked stimulation of activity, especially at low Ca2+ concentrations, but the Ka for Ca2+ was shifted only slightly, to 2.5 microM. With histone as substrate, the enzyme had a very high Ka (greater than 50 microM) for Ca2+, which was substantially decreased to 3 microM-Ca2+ by diolein. A Triton X-100 mixed-micelle preparation of lipids was also utilized to assay protein kinase C with histone as the substrate. Under these conditions kinase activity was almost totally dependent on the presence of diolein; again, diolein caused a large decrease in the Ka for Ca2+, from greater than 100 microM to 2.5 microM. The increased sensitivity of protein kinase C to Ca2+ with P47 rather than histone, and the ability of diacylglycerol to activate protein kinase C without shifting the Ka for Ca2+, when P47 is the substrate, illustrate that the mechanism of protein kinase C activation is influenced by the exogenous substrate used to assay the enzyme.     

Synthetic peptide substrates for a tyrosine protein kinase

Immunoprecipitates containing the transforming protein of the avian sarcoma virus, Y73, together with its associated tyrosine-specific protein kinase, have an activity which will phosphorylate the synthetic peptide Lys-Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg at the tyrosine residue. This peptide corresponds to 10 out of 11 amino acids surrounding the phosphorylated tyrosine in both pp60src and P90, the transforming proteins of Rous sarcoma virus and Y73 virus, respectively. The apparent Km for phosphorylation of the peptide was about 5 mM. A second peptide with the sequence Lys-Leu-Ile-Asp-Asn-Glu-Tyr-Thr-ala-Arg differing from the first peptide only by the absence of the glutamic acid 4 residues from the tyrosine was also phosphorylated, but the apparent Km for the reaction was 40 mM. Several sites of tyrosine phosphorylation in viral transforming proteins have been found to have one or more glutamic acids close to the phosphorylated tyrosine on the NH2-terminal side. Taken together with our in vitro phosphorylation studies, this suggests that the primary sequence surrounding target tyrosines may play a role in recognition of substrates by tyrosine protein kinases, and in particular, that glutamic acid residues on the NH2-terminal side may be important.     

Binding of protein kinase substrates by fluorescently labeled calmodulin

A synthetic protein kinase substrate, PRO-LEU-SER-ARG-THR-LEU-SER-VAL-SER-SER-NH₂, undergoes calcium-dependent binding by calmodulin. Phosphorylation of the peptide decreases its affinity for calmodulin with the dissociation constant increasing from 2.4 to $\text{ca}$. 7 mM. The results are consistent with the suggestion that calmodulin and the cAMP-dependent protein kinase can act on common recognition sequences.     

The wheat germ cell-free based screening of protein substrates of calcium/calmodulin-dependent protein kinase II delta

Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in mediating calcium signaling. Here, we demonstrate a method for screening substrates phosphorylated by human CaMKII delta using a wheat cell-free system. The cell-free mixture expressing CaMKII delta was incubated with HeLa extracts and radiolabeled ATP. From analysis of two-dimensional electrophoresis gels and mass spectrometry, two proteins were found. The cell-free based in vitro kinase assay revealed that CaMKII delta phosphorylates eukaryotic translation initiation factor 4B and stress-induced phosphoprotein 1 (STIP1), the latter on Ser189. Furthermore, constitutively-active CaMKII delta phosphorylated STIP1 in HeLa cells and dramatically promoted nuclear localization of STIP1, suggesting that calcium signals via CaMKII delta may regulate subcellular localization of STIP1. This approach may be a useful tool for target screening of protein kinases.