Communication between individual cells in multicellular organisms is essential for the regulation and coordination of complex cellular processes such as growth, differentiation, migration and apoptosis. Signal transduction networks mediating these biological mechanisms use reversible protein phosphorylation as a fundamental tool. According to a modest estimation about one third of the known proteins can be phosphorylated in eukaryots. In the past decades it turned out that a multitude of regulatory cycles are intimately interlocked through overlapping substrates and the mutual regulation of kinases and phosphatases via phosphorylation/dephosphorylation by other kinases and phosphatases, respectively (Cohen, P. (2000) Trends Biochem. Sci. 25, 596-601). Deregulation of enzymatic dephosphorylation will cause substantial malfunctions and therefore are causal for diseases.
Protein phosphatases are grouped according to their activity on the various phospho-amino acids, namely the most prominent phosphorylated amino acids serine (Ser), threonine (Thr) and tyrosine (Tyr). However, further amino acids need to be considered as relevant phospho-derivatives, namely histidine (His), lysine (Lys) cysteine (Cys), aspartic acid (Asp), and glutamic acid (Glu). Systematic classification of kinases concern the nature of the phosphate accepting amino acids, e.g. whether a phosphate ester, thio ester, phospho-amidate or phosphate carboxylate anhydride is formed (Matthews, H. R. & Chan, K. (2001) Methods Mol. Biol. 124: 171-182).
The phospho-amino acids His, Asp, Glu, Lys and Cys show distinct chemical properties like acid-lability and faster physiological turnover which are commonly used for characterization (Sickmann, A. & Meyer, H. E. (2001) Proteomics 1, 200-206). Up to now the less abundant phospho-amino acids predominantly showed up in cellular processes of bacteria, plants (Chang, C. & Meyerowitz, E. M. (1995) Proc. Natl. Acad. Sci. USA 92, 4129-4133) and lower eucaryots (Huang, J. M. et al. (1991) J. Biol. Chem. 266, 9023-9031). Due to their high-energy phosphate bonds they are utilized in rapid phosphotransfer reactions like the two-component system and the phospho-relay signaling (West, A. H. & Stock, A. M. (2001) Trends Biochem. Sci. 26, 369-376).
Most recently, histidine phosphorylation was identified to contribute to regulatory processes in vertebrates like stimulation of human platelets (Crovello, C. S. et al. (1995) Cell 82, 279-286) or regulating annexin I-function (Muimo, R. et al. (2000) J. Biol. Chem. 275, 36632-36636). Another well established example is the regulative phosphorylation of ATP-citrate lyase by nucleoside diphosphate kinase. (NDPK) (Wagner, P. D. & Vu, N. D. (1995) J. Biol. Chem. 270, 21758-21764). However, protein phosphoamidases acting specific on phospho-amidates in proteins like histidine phosphorylated proteins are rarely described. First evidence for such like phosphoamidase activity was found as additional function of PP1 and PP2A when acting on P-His histone 4 (Kim, Y. et al. (1993) J. Biol. Chem. 268, 18513-18518; Kim, Y. et al. (1995) Biochim. Biophys. Acta 1268, 221-228). Although histidine phosphate is known to be present in mammals (Crovello, C. S. et al. (1995) Cell, 82:279-286), it has not to date been possible to identify either the corresponding kinases or the relevant phosphoamidases. A protein with assigned specific phosphoamidase activity, recognizing amongst other substrates P-His-NDPK but not hydrolyzing O-phosphorylated substrates, was isolated from bovine liver but no sequence identity is published (Hiraishi, H. et al. (1999) J. Biochem. (Tokyo) 126, 368-374). Furthermore, a 17-kDa phosphoamidase that is specific only for phosphorylated Arg and is free of the activity that hydrolyzes O-phosphorylated compounds has been described (Kuba, M. et al. (1992) Eur. J. Biochem. 208, 747-752; Yokoyama, K., et al. (1993) J. Biochem. 113, 236-240 Kumon, A. et al. (1996) J. Biochem. 119, 719-724).
A protein phosphoamidase specific for hydrolyzing N-phosphorylated histidine residues in peptides or proteins and having no activity that hydrolyzes O-phosphorylated peptides or proteins is PHP1 (WO 00/52175; Ek, P. et al. (2002) Eur. J. Biochem. 269, 5016-5023).
In scope to understand the physiological role of phosphoamidases and to be able to interact with patho-physiological situations it is important to identify the enzyme-substrate specificity of a phosphoamidase unequivocally, and to develop tools to interfere by means of an inhibition or activation of the enzyme.
Within the meaning of the present invention the term “phosphoamidase” defines an enzyme hydrolyzing phosphoamide (P—N) bonds of phosphorylated basic amino acids like P-His, P-Lys, P-Arg, or of peptides or proteins comprising these phosphorylated amino acids and which is devoid of an activity that hydrolyzes O-phosphorylated proteins or peptides. Examples for such phosphoamidases within the meaning of the present invention are described in Hiraishi, H. et al. (1999) J. Biochem. (Tokyo) 126, 368-374, Kuba, M. et al. (1992) Eur. J. Biochem. 208, 747-752 and WO 00/52175.
Within the meaning of the present invention the term “protein phosphoamidase” defines a phosphoamidase hydrolyzing phosphoamide (P—N) bonds of phosphorylated basic amino acids like P-His, P-Lys or P-Arg only within peptides or proteins and which is devoid of an activity that hydrolyzes O-phosphorylated proteins or peptides. An example for such a protein phosphoamidase within the meaning of the present invention is the protein histidine-phosphate specific phosphoamidase PHP1 described in WO 00/52175.
Within the meaning of the present invention the term “protein histidine-phosphoamidase” defines a protein phosphoamidase specific for hydrolyzing phosphorylated histidine within peptides or proteins. An example within the meaning of the present invention is PHP1 as described in WO 00/52175.
Within the meaning of the present invention the term phosphatase defines an enzyme hydrolyzing phosphoester (P—O) bonds of phosphorylated amino acids like P-Thr, P-Ser or P-Tyr, or of peptides or proteins comprising these phosphorylated amino acids. These enzymes may have additionally a phosphoamidase activity.
Within the meaning of the present invention the term “protein phosphatase” defines a phosphatase hydrolyzing phosphoester (P—O) bonds of phosphorylated amino acids like P-Thr, P-Ser or P-Tyr only within peptides or proteins. These enzymes may have additionally a phosphoamidase activity. Examples for protein phosphatases within the meaning of the present invention are PP1, PP2A and PP2C (Kim, Y. et al. (1993) J. Biol. Chem. 268, 18513-18518).
Within the meaning of the present invention the term “phosphoamidase activity” defines the hydrolyzation of a phosphoamide bond irrespective whether it is hydrolyzed by a phosphoamidase or phosphatase.
Within the meaning of the present invention the term “activity of a phosphoamidase” defines the hydrolyzation of a phosphoamide bond by a phosphoamidase, protein phosphoamidase or protein histidine phosphoamidase which is devoid of an activity that hydrolyzes phosphoester (P—O) bonds of phosphorylated proteins or peptides.
There is some literature on biochemical methods suitable for detection of phosphohistidine dephosphorylation. For example the malachite green method (Ohmori, H. et al. (1993) J. Biol. Chem. 268, 7625-7627; Ohmori, H. et al. (1994) J. Biochem. 116, 380-385) has been used to identify phosphoamidase activity for 6-phospholysine and 3-phosphohistidine. Released Pi was assayed successive addition of malachite green and citric acid. The absorbance of the phosphate/molybdate complex was measured at 630 nm (Kuba, M. et al. (1992) Eur. J. Biochem. 208, 747-752). However this assay may strongly interfere with the acid lability of physiological phosphoamidase substrates. 6-phospholysine, 3-phosphohistidine and the corresponding dephosphorylated amino acids have been identified by thin layer electrophoresis on cellulose and staining with ninhydrin or Rosenberg's reagent.
Using 32P-labeled histidine-phosphorylated histone H4, the protein phosphatases PP1, PP2A and PP2C have been shown to have a protein phosphoamidase activity for histidine in addition to their Ser-, Thr- or Tyr-phosphatase activity (Kim, Y. et al. (1993) J. Biol. Chem. 268, 18513-18518; Matthews, H. R. & MacKintosh, C. (1995) FEBS Lett. 364, 51-54; Kim, Y. et al. (1995) Biochim. Biophys. Acta 1268 221-228). Phosphoamidase activity for histidine was detected by incubation of [32P]histone 4 with phosphatase and subsequent acrylamide-electrophoresis of the reaction mixture and autoradigraphy of the gel. For quantitative analysis 32Pi released from [32P]histone 4 was removed by ultracentifugation, and the remaining histone-bound phosphate was quantified by liquid scintillation counting.
A further test on the activity of a protein His-phosphoamidase has been described in WO 00/52175 using 32P-labeled histidine-phosphorylated CheA as substrate. CheA is a recombinant bacterial histidine autokinase (Bilwes, A. M. et al. (1999) Cell 96, 131-134). Free phosphate has been identified by thin-layer chromatography via ammonium molybdate or by autoradiography, respectively.
A further possibility to detect inorganic phosphate has been described in WO 95/02825 and Brune et al. 1994 (Biochemistry (1994) 33, 8262-8271). This assay is based on the rapid shift in the fluorescence characteristics of MDCC-PBP, which is the E. coli phosphate binding protein (PBP) labelled with N-[2-(1)-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), a detectable fluorescent label.
However, because the methods mentioned above can not be used in a continuous assay, or are time consuming and/or generate radioactive waste, they are not well applicable in HTS (High Throughput Screening) runs. Therefore there is a need of further protein phosphoamidase and phosphoamidase assays which are easy to perform and applicable in HTS runs.
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