PTKs are involved in many cell signaling pathways. These enzymes represent early key regulators in such functions as cell growth, proliferation, differentiation, secretion, and apoptosis [1Gschwind A, Fischer OM, Ullrich A. The discovery of receptor tyrosine kinases: targets for cancer therapy Nat Rev Cancer 2004; 4(5 ): 361-70.]. Aberrant protein kinase expression or function is a cause or consequence of many human diseases like cancer and atherosclerosis. As a result, kinases represent very important targets in the development of therapeutics.

Protein kinase selectivity is critical for appropriate intracellular signaling. Kinase-substrate interactions are mediated via specific interaction sites such as SH2 domains, but also by the distinct specificity of the catalytic domain. The use of small peptides to investigate kinase specificity and catalysis is supported by crystallographic structures of kinase-peptide complexes as well as studies with synthetic peptide libraries [2Hubbard SR. Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog EMBO J 1997; 16(18 ): 5572-81.,3Songyang Z, Carraway KL, Eck MJ, et al. Catalytic specificity of protein-tyrosine kinases is critical for selective signalling Nature 1995; 373(6514 ): 536-9.]. Considering that almost all human PTKs have recently become available as purified recombinant enzymes, it has become possible to profile peptide substrate activity throughout the entire group to possibly reveal novel specificities of individual kinases and kinase sub-families. Previously, a study was performed on the yeast kinome [4Zhu H, Klemic JF, Chang S, et al. Analysis of yeast protein kinases using protein chips Nat Genet 2000; 26(3 ): 283-9.] in which nearly all (119) of the yeast protein kinases were overexpressed and assayed in a high-throughput manner on 17 chip-immobilized protein and peptide substrates. However, such an extensive investigation has not been performed with human PTKs.

In this study, three different peptides previously characterized with a small subset of PTK(s) were chosen for screening 81 human PTKs. The three peptides are derived from bona fide kinase substrate sequences and therefore represent putative in vivo targets. The CDK1 (Tyr15) or p34^cdc2 (Tyr15) peptide was originally shown to be a suitable substrate for four Src kinases: SRC, LYN, LCK, and FYN [5Cheng HC, Nishio H, Hatase O, Ralph S, Wang JH. A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases J Biol Chem 1992; 267(13 ): 9248-56.]. The IRS1 (Tyr983) peptide was chosen to represent a YMXM peptide. Peptides containing the YMXM motif were shown as effective substrates for receptor tyrosine kinases such as the insulin receptor [6Shoelson SE, Chatterjee S, Chaudhuri M, White MF. YMXM motifs of IRS-1 define substrate specificity of the insulin receptor kinase Proc Natl Acad Sci USA 1992; 89(6 ): 2027-31.], and also for non-receptor Tyr kinases [7Garcia P, Shoelson SE, George ST, Hinds DA, Goldberg AR, Miller WT. Phosphorylation of synthetic peptides containing Tyr-Met-X-Met motifs by nonreceptor tyrosine kinases in vitro J Biol Chem 1993; 268(33 ): 25146-51.]. Finally, the JAK1 (Tyr1023) peptide was shown to be a substrate for JAK3 [8Wang R, Griffin PR, Small EC, Thompson JE. Mechanism of Janus kinase 3-catalyzed phosphorylation of a Janus kinase 1 activation loop peptide Arch Biochem Biophys 2003; 410(1 ): 7-15.]. In addition to the small peptides, the generic PTK substrates polyGT and polyGAT [9Braun S, Raymond WE, Racker E. Synthetic tyrosine polymers as substrates and inhibitors of tyrosine-specific protein kinases J Biol Chem 1984; 259(4 ): 2051-4.] were tested as controls of PTK activity.

The high-throughput TR-FRET platform, LANCE^®Ultra, was used to measure enzyme activity, whereby the phosphopeptide product is detected by a highly sensitive anti-pTyr antibody labeled with europium chelate (Fig. 1). TR-FRET occurs between the chelate-labeled antibody and the dye-conjugated phosphopeptide upon excitation at 320 or 34 nm.

Fig. (1). LANCE Ultra TR-FRET technology for kinase assays In these kinase assays, the binding of a Eu-labeled anti-phospho- Tyr antibody (Eu-Ab) to the phosphorylated ULight-labeled substrate brings donor and acceptor molecules into close proximity. After irradiation of the kinase reaction at 320 or 340 nm, the energy from the Eu donor is transferred to the ULight acceptor dye which, in turn, generates light at 665 nm. The intensity of the light emission is proportional to the level of ULight-substrate phosphorylation.

Apart from providing valuable activity data for the 81 PTKs, the profiling experiments demonstrated that the CDK1 and IRS1 peptides are highly selective for certain kinase sub-families, whereas the polyGT, polyGAT, and JAK1 peptides are promiscuous. Furthermore, novel activities for certain kinases, such as the Eph family, have been observed.

In the present work, almost all of the human PTKs were assayed with five peptidic substrates using a TR-FRET technology and purified recombinant enzymes. A unique pattern of human PTK selectivity was observed for the CDK1 and IRS1 peptides, whereas polyGT and polyGAT were substrates for almost all of the kinases and JAK1 was active with over 60% of the kinases. In order to visually demonstrate the selectivity of the peptide substrates, the activity of the PTKs towards the JAK1, CDK1, and IRS1 peptides is illustrated in dendrogram format in Fig. (3). In contrast to the JAK1 peptide, where almost all branches are highlighted with active kinases, the CDK1 and IRS1 peptides are only substrates for certain sub-families.

Fig. (3). Dendrograms of PTK peptidic substrate selectivity Kinases with S/B ratios greater than 3 are highlighted. The names of the kinases not tested were faded out. Dendrogram illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).

It was clearly observed that, indeed, polyGT and polyGAT can be considered as universal substrates for Tyr kinases and serve as good positive controls for PTK activity. On the other hand, using these non-specific substrates may be a disadvantage, because contaminating Tyr kinases may generate a signal that is not due to the kinase being studied. Polymeric peptides containing acidic amino acids and Tyr were originally described about 25 years ago [9Braun S, Raymond WE, Racker E. Synthetic tyrosine polymers as substrates and inhibitors of tyrosine-specific protein kinases J Biol Chem 1984; 259(4 ): 2051-4.]. To our knowledge, this is the first time that these polymeric peptides have been tested with such a wide variety of Tyr kinases.

Interestingly, the JAK1 peptide seemed to be promiscuous for many Tyr kinases and it also represented a better substrate than polyGT and polyGAT in some cases. This broad activity may be related to the fact that it contains three acidic residues proximal to the Tyr residues: ETDKEYY. As for the polyGT and polyGAT polymers, the presence of acidic amino acids may cause the JAK1 peptide to be a non-specific substrate for Tyr kinases. The lack of specificity towards the JAK1 peptide may also be a caution in other studies of peptide substrate specificity of PTKs. PTK binding to acidic peptides may be unrelated to the specificity of the kinase, but to a general structural preference of all the PTKs.

In the current study, all Src family kinases efficiently utilized the CDK1 peptide as a substrate and further demonstrates the overall preference of the Src family for the CDK1 peptide [5Cheng HC, Nishio H, Hatase O, Ralph S, Wang JH. A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases J Biol Chem 1992; 267(13 ): 9248-56.]. Interestingly, two of the three members of the Src-related Frk family, BRK and FRK, do not phosphorylate the CDK1 peptide, although both phosphorylated polyGT and polyGAT. Although the Frk family of kinases has sequence homology to the Src family, the catalytic specificities of the two families are different, further demonstrating the distinct nature of the Frk family of kinases [12Serfas MS, Tyner AL. Brk, Srm, Frk, and Src42A form a distinct family of intracellular Src-like tyrosine kinases Oncol Res 2003; 13(6-10 ): 409-19.].

The ability of almost all Eph kinases to phosphorylate the CDK1 peptide was not predicted. A single report implicating EphA5 as a putative kinase phosphorylating CDK5 Tyr15 has been published [13Cheng Q, Sasaki Y, Shoji M, et al. Cdk5/p35 and Rho-kinase mediate ephrin-A5-induced signaling in retinal ganglion cells Mol Cell Neurosci 2003; 24(3 ): 632-45.], and the preference of the Eph family towards this substrate was not revealed by previous studies using synthetic peptides against the EphA3 and EphA4 kinases [14Warner N, Wybenga-Groot LE, Pawson T. Analysis of EphA4 receptor tyrosine kinase substrate specificity using peptide-based arrays FEBS J 2008; 275(10 ): 2561-73.,15Davis TL, Walker JR, Allali-Hassani A, Parker SA, Turk BE, Dhe-Paganon S. Structural recognition of an optimized substrate for the ephrin family of receptor tyrosine kinases FEBS J 2009; 276(16 ): 4395-04.]. However, the sequence around Tyr15 in CDK5, KIGEGTYGTVFK, is very similar to the CDK1 peptide in this study: KIGEGTYGVVYK. This is the first time that the Eph family has been shown to directly phosphorylate the CDK-1 (Tyr-15) sequence. The Eph family of receptors is of particular importance due to their role in communication between adjacent cells and there are strong efforts to understand the complex signaling mechanisms of these PTKs [16Pasquale EB. Eph-ephrin bidirectional signaling in physiology and disease Cell 2008; 133(1 ): 38-52.].

In the current study, 26 of the 32 kinases found to be active against the IRS1 peptide were receptor Tyr kinases, demonstrating that the IRS1 peptide is greatly favored by receptor Tyr kinases. Also, the IRS1 peptide was a very good substrate for JAK-1, 2, and 3 kinases, which are cytoplasmic kinases. The activity of the JAK kinases towards IRS1 has been previously reported [17Yin T, Keller SR, Quelle FW, et al. Interleukin-9 induces tyrosine phosphorylation of insulin receptor substrate-1 via JAK tyrosine kinases J Biol Chem 1995; 270(35 ): 20497-502.]. Moreover, Jak family kinases show more sequence homology to receptor Tyr kinases than to cytoplasmic Tyr kinases. Indeed, the kinases acting on the IRS1 peptide tend to group entirely on the left side of the dendrogram shown in Fig. (3).

Overall, a high-throughput assay technology was used to profile five Tyr kinase substrates. The CDK1 and IRS1 peptides demonstrated high selectivity for certain subfamilies, whereas the JAK1 peptide is promiscuous. Furthermore, this is the first publication showing the specificity of the Eph family towards the CDK1 (Tyr-15) sequence. Researchers may refer to this study to identify possible substrates for their kinase(s) of interest or to set-up kinase assays to further study the structure and function of the PTK(s).

None Declared.