still remain. During the preparation of the present manuscript, Srivastava et al. (11) published the finding that the calcium-activated K channel KCa3.1, phosphorylated on a histidine residue by nucleoside diphosphate kinase beta (NDPK-B), was dephosphorylated by PHPT1 which caused a de- crease in the K channel activity in CD4 T cells. However, whether KCa3.1 is a main physiological substrate of PHPT1, or only one of several physio- logical substrates, remains to be established. In the present work, we selected an immunohis- tochemical approach in an attempt to further characterize the role of PHPT1. A screening of a large number of tissues for the localization of the enzyme protein was considered to give independent hints as to the function of the enzyme. Such a study also made possible the comparison of the mRNA expression (7) and the protein expression of PHPT1. PHPT1 protein was expressed in all organs tested, even though the extent varied. In order to eliminate possible mistakes in the interpretation of results, the specificity of the primary antibody was carefully tested using both preimmune serum and depletion assays as controls. This confirmed that the antibodies were specific, since no staining for PHPT1 was seen after the antibody had been saturated with the PHPT1 peptide 101 115 prior to being added to the tissue (Figure 2D) or when preimmune serum was used (Figure 1B). There was a fairly good correlation between protein expression and the expression level of mRNA. Similar results are found in the PubMed UniGene ESTProfile database. It must be empha- sized, however, that the comparison was made between organ-specific cell expression of PHPT1 protein and whole-organ expression of PHPT1 mRNA. Protein kinases and phosphatases are often involved in cell proliferation. It is therefore note- worthy that PHPT1 expression was strong in varying types of rapidly dividing epithelial tissues but not detectable in most of the endothelium, a normally quiescent epithelium, in both species tested and that all human cancers of ectodermal origin showed moderate PHPT1 expression (HPR homepage). Another example of PHPT1 being involved in a proliferation context is a recurrent 9q34 duplication in paediatric T cell acute lymphoblastic leukaemia that was found in 33% of the cases with increased mRNA expression of the PHPT1 gene (12). In the HPR database, the relative PHPT1 protein expres- sion level was moderate, 8%, in the only human leukaemia cell line analysed, but the presence or absence of duplication in this particular cell line is not known. Further, the expression of PHPT1 is Immunohistochemical localization of PHPT1 71 increased upon ex vivo radiation exposure of blood cells, as are CDKN1A, FDXR, SESN1, and BBC3, known possible regulators in cell proliferation or apoptosis (13). Additionally, in the PubMed Uni- Gene ESTProfile database, glioma cells have high expression of PHPT1 mRNA, which correlates to the HPR finding for malignant glioma and glioblas- toma cell lines, where the immunostaining was moderate to strong. Normal glia cells in both adult mouse and human did not express PHPT1. In view of these findings we suggest that PHPT1 might have a role during cell proliferation and differentiation. The results from the immunohistochemical staining of PHPT1 in human tissues were essentially similar to mouse PHPT1 expression patterns. The strongest signal was found in the squamous epithelial cells of skin, oesophagus, vagina, and cervix. How- ever, some tissues showed expression level differ- ences between the two species or even contradictory results. The reasons for these differences are not clear. They may be due to the differences in tissue preparation or other methodological differences between the laboratories. A partial homology to the sex-regulated protein Jan A in Drosophilae prompted us to investigate the expression of PHPT1 in the reproductive organs. The protein had a moderate level of expression in both human and mouse testis and in the human uterus epithelium (not determined in mouse). In the PubMed UniGene ESTProfile database, mRNA expression is high also in the mammary gland, but in this database there are also organs that have even higher levels of mRNA expression, e.g. heart, pituitary gland, and parathyroid glands. A specific role for the enzyme in sex regulation is therefore not obvious. The staining profile of ATP-citrate lyase, re- ported to be a substrate of PHPT1 (10), did not overlap that of PHPT1 in human normal tissue, cancer cells, and cell lines, as can be seen on the HPR home page. The beta subunit of G protein, reported to be colocalized with and dephosphory- lated by PHPT1 (9), is not represented in the HPR database and can therefore not be compared with the staining profile of PHPT1. This is also the case for the calcium-activated K channel KCa3.1 Since this channel and PHPT1 could be coimmunopreci- pitated in the CD4 T cells (11), the localization of at least a part of the PHPT1 protein, as studied in the present work, may coincide with that of KCa3.1. However, the resolution of the immunohistochem- ical method used does not allow for an unequivocal discrimination between a PHPT1 signal from the plasma membrane and from other subcellular struc- tures except for a clear expression in nuclei found for