Histidine kinases and the missing phosphoproteome from prokaryotes to eukaryotes

Abstract

Protein phosphorylation is the most common type of post-translational modification in eukaryotes. The phosphoproteome is defined as the complete set of experimentally detectable phosphorylation sites present in a cell's proteome under various conditions. However, we are still far from identifying all the phosphorylation sites in a cell mainly due to the lack of information about phosphorylation events involving residues other than Ser, Thr and Tyr. Four types of phosphate-protein linkage exist and these generate nine different phosphoresidues-pSer, pThr, pTyr, pHis, pLys, pArg, pAsp, pGlu and pCys. Most of the effort in studying protein phosphorylation has been focused on Ser, Thr and Tyr phosphorylation. The recent development of 1- and 3-pHis monoclonal antibodies promises to increase our understanding of His phosphorylation and the kinases and phosphatases involved. Several His kinases are well defined in prokaryotes, especially those involved in two-component system (TCS) signaling. However, in higher eukaryotes, NM23, a protein originally characterized as a nucleoside diphosphate kinase, is the only characterized protein-histidine kinase. This ubiquitous and conserved His kinase autophosphorylates its active site His, and transfers this phosphate either onto a nucleoside diphosphate or onto a protein His residue. Studies of NM23 protein targets using newly developed anti-pHis antibodies will surely help illuminate the elusive His phosphorylation-based signaling pathways. This review discusses the role that the NM23/NME/NDPK phosphotransferase has, how the addition of the pHis phosphoproteome will expand the phosphoproteome and make His phosphorylation part of the global phosphorylation world. It also summarizes why our understanding of phosphorylation is still largely restricted to the acid stable phosphoproteome, and highlights the study of NM23 histidine kinase as an entrée into the world of histidine phosphorylation.

Fig. 1

Chronology of phosphorylation discovery. Black circles with upper legends correspond to major events in histidine phosphorylation. For reference; 1883–1906 ^,, 1951–56 ^,, 1962 , 1980 , 1981 ^,, 1995 , 2000 , 2010 , 2015 .

Fig. 2

Overall phosphorylation and phosphate linkages. Phosphorylases and kinases are distinguished by the source of donor phosphate. Phosphorylases add a phosphate group (OP) to an acceptor (A) breaking an A–B bond using inorganic phosphate (H-OP), e.g. glycogen phosphorylase, whereas kinases transfer a phosphate group (P) from a donor (B) (usually ATP) to an acceptor (A). SONAtes for S-O-N-A-phosphate categories: S-phosphate (Thiophosphate), O-phosphate (Phosphate-ester), N-phosphate (Phosphoramidate) and A-phosphate (Acyl-phosphate group).

Fig. 3

Two-component system for autophosphorylation of a transmembrane histidine kinase (HK). A sensor perceives an extracellular stimulus. The substrate for the His kinase is the response regulator, which becomes phosphorylated on a specific Asp residue. In bacteria, the response regulator generally has a common domain with at least two Asp and one Lys residue . HK receptors are homodimeric proteins, and both trans and cis phosphorylations have been reported.

Fig. 4

Temperature/pH compatibility in function of phosphoresidue. A. Temperature is inversely correlated to pH gradient for pHis stability. The converse is observed for pSer/Thr residues, whereas pTyr residues are highly stable over the whole pH range. This representation is designed to indicate the recommended experimental conditions to conserve different types of phosphorylation but needs to be adapted according to the experimental buffer solution used. In addition, stability can also be impacted by salt conditions, tertiary structure and neighboring amino acids. Black stars on pH gradient represent different human physiological states. B. A dot blot of recombinant pNM23-1/2 H118 is shown in the upper right hand panel, using purified NM23-H1 (according to Fuhs et al. ), either unphosphorylated, or autophosphorylated in the presence of ATP, or autophosphorylated in the presence of ATP followed by boiling followed by incubation with pre-immune serum (PI 95) or antibodies purified on immobilized antigen peptide from serum raised against a peptide representing residues 114–122 of NM23-1/2 containing 1-pTza in place of His118. The non-hydrolyzable 1-pTza NM23-1/2 peptide sequence analogue for H118 is used as a control. C. Immunoblots of SDS gel-fractionated lysates of HeLa cells or ALVA-31 prostate cancer cells with anti-pNM23-1/2 antibodies (top), a mixture of anti-1-pHis and 3-pHis monoclonal antibodies (middle) in the lower right hand panel, comparing conditions where pHis is stable (pH 10, 4°C) or where it is unstable (pH 3 (0.1% formic acid CH₂O₂), 90°C for 5 min, restored to pH 8.8 with 1 M Tris). The parenthesis indicates the pNM23-1/2 bands, and the arrow indicates a heat-insensitive band that is apparently recognized non-specifically by the purified antibodies. The levels of NM23-H1/2 and actin in each sample were determined by immunoblotting with specific antibodies (lower panels).