Identification and characterization of a mammalian 14‐kDa phosphohistidine phosphatase

Abstract

Protein histidine phosphorylation in eukaryotes has been sparsely studied compared to protein serine/threonine and tyrosine phosphorylation. In an attempt to rectify this by probing porcine liver cytosol with the phosphohistidinecontaining peptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide (phosphopeptide I), we observed a phosphatase activity that was insensitive towards okadaic acid and EDTA. This suggested the existence of a phosphohistidine phosphatase different from protein phosphatase 1, 2A and 2C. A 1000-fold purification to apparent homogeneity gave a 14-kDa phosphatase with a specific activity of 3 lmolAEmin )1 AEmg )1 at pH 7.5 with 7 lM phosphopeptide I as substrate. Partial amino-acid sequence determination of the purified porcine enzyme by MS revealed similarity with a human sequence representing a human chromosome 9 gene of hitherto unknown function. Molecular cloning from a human embryonic kidney cell cDNAlibrary followed by expression and purification, yielded a protein with a molecular mass of 13 700 Da, and an EDTA-insensitive phosphohistidine phosphatase activity of 9 lmolAEmin )1 AEmg )1 towards phosphopeptide I. No detectable activity was obtained towards a set of phosphoserine-, phosphothreonine-, and phosphotyrosine peptides. Northern blot analysis indicated that the human phosphohistidine phosphatase mRNA was present preferentially in heart and skeletal muscle. These results provide a new tool for studying eukaryotic histidine phosphorylation/dephosphorylation.Keywords: dephosphorylation; N-phosphorylation; phosphoamidase; phosphopeptide; protein histidine phosphatase.Boyer and coworkers detected protein-bound phosphohistidine in rat-liver mitochondrial succinyl-CoA synthetase almost 40 years ago [1,2]. Despite the long time interval and the fact that phosphohistidine represents a substantial fraction of eukaryotic protein-bound phosphate [3], only a few phosphohistidine-containing proteins have been detected compared to the large number of eukaryotic proteins phosphorylated on serine, threonine and tyrosine residues. One reason for this difference may be that the N-bound phosphate of phosphohistidine easily escapes detection by common analytical procedures, due to its lability under acidic conditions, e.g. during fixation and staining of gels after SDS/PAGE [4].The studies on eukaryotic protein histidine phosphorylation and dephosphorylation have dealt with essentially two aspects. One is the intermediary phosphorylation of enzymes [5][6][7][8][9][10], of which nucleoside diphosphate kinase is a particularly well-studied example. The other is the reversible protein histidine phosphorylation by protein kinases and phosphatases [3,11]. An important contribution to the latter field was the purification of a yeast protein histidine kinase in 1991 [12]. Access to this enzyme also made possible the preparation of 32 P-labelled histone H4, which was later used as substrate in the search for phosphohistidine phosphatases. Using such an approach, the catalytic subunits of the well-studied serine/...

p53-directed translational control can shape and expand the universe of p53 target genes

“…We found 55 translatome-uncoupled DEGs common to the two treatments ( Figure 2a). Among them, PHPT1 (14-kDa phosphohistidine phosphatase) 22 and TP53RK (p53-related protein kinase) 23 were chosen for validation by qPCR owing to their biological relevance in post-translational control, and because they had not been previously reported as p53-regulated genes. PHPT1 and TP53RK proved to be translational upregulated genes, particularly after 16 h of treatment ( Figure 2b).…”

“…Guided by the comparison of transcriptome and translatome data, but also considering DEGs within the free cytoplasmic pool (sub), we identified a number of RBPs that could be regulated by either Doxo (67 RBPs) or nutlin-3a (30 RBPs) or would be common to both treatments (22) (Supplementary Table S4). These 22RBPs are primary candidates for p53-directed control.…”

Advances in development of new tools for the study of phosphohistidine

“…28 In this particular case, it is not clear which isomer of pHis is formed and on which His residue. Mammalian pHis phosphatases, which have been characterized include: protein pHis phosphatase 1 (PHPT1); 17,[29][30][31][32][33][34][35] Lys/His phosphatase (LHPPase); 36,37 Ser Thr protein phosphatases (PP1/2 A/2C); 38,39 T-cell ubiquitin ligand-2 (TULA-2); 40,41 and the recently reported phosphoglycerate mutase-5 (PGAM5). 42 In addition, pHis phosphatase activity has been reported in rat tissue extracts but these have not been fully characterized.…”

14-kDa Phosphohistidine phosphatase plays an important role in hepatocellular carcinoma cell proliferation

“…Advances in HCC treatment are likely to require a more detailed understanding of its biology and behavior; thus, the mechanisms underlying the rapid metastatic capacity of HCC require further study (10,11). The 14-kDa phosphohistidine phosphatase (PHP14), also known as PHPT1, was the first histidine phosphatase protein identified in vertebrates and is similar to the janus proteins of Drosophila (12,13). PHPT1 demonstrates specific phosphatase activity against peptides and proteins containing phosphohistidine (14).…”