The neuropeptide processing enzyme EC 3.4.24.15 is modulated by protein kinase A phosphorylation.

The metalloendopeptidase EC (EP24.15) is a neuropeptide-metabolizing enzyme expressed predominantly in brain, pituitary, and testis, and is implicated in several physiological processes and diseases. Multiple putative phosphorylation sites in the primary sequence led us to investigate whether phosphorylation effects the specificity and/or the kinetics of substrate cleavage. Only protein kinase A (PKA) treatment resulted in serine phosphorylation with a stoichiometry of 1.11 +/- 0.12 mol of phosphate/mol of recombinant rat EP24.15. Mutation analysis of each putative PKA site, in vitro phosphorylation, and phosphopeptide mapping indicated serine 644 as the phosphorylation site. Phosphorylation effects on catalytic activity were assessed using physiological (GnRH, GnRH(1-9), bradykinin, and neurotensin) and fluorimetric (MCA-PLGPDL-Dnp and orthoaminobenzoyl-GGFLRRV-Dnp-edn) substrates. The most dramatic change upon PKA phosphorylation was a substrate-specific, 7-fold increase in both K(m) and k(cat) for GnRH. In both rat PC12 and mouse AtT-20 cells, EP24.15 was serine-phosphorylated, and EP24.15 phosphate incorporation was enhanced by forskolin treatment, and attenuated by H89, consistent with PKA-mediated phosphorylation. Cloning of the full-length mouse EP24.15 cDNA revealed 96.7% amino acid identity to the rat sequence, and conservation at serine 644, consistent with its putative functional role. Therefore, PKA phosphorylation is suggested to play a regulatory role in EP24.15 enzyme activity.

PAR, a novel androgen regulated gene, ubiquitously expressed in normal and malignant cells.

During our work on the mechanism of hormone resistance of prostatic carcinomas, a novel gene that we called PAR (prostate androgen regulated) was isolated from an androgen resistant subline (LNCaP-OM) using a modified representational difference analysis. The complete sequence of the gene cDNA has 1029 nucleotides with a continuous reading frame of 438 bases encoding for 146 amino acids. Its deduced amino acid sequence has motifs for myristoylation and phosphorylation by protein kinase C. The PAR gene was overexpressed in all prostatic carcinoma cell lines studied (LNCaP, DU145, PC3 and LNCaP-OM) compared to the normal prostatic tissue. Furthermore, its expression was higher in androgen resistant prostate cancer lines DU145, PC3 and LNCaP-OM, in comparison to androgen sensitive LNCaP cells. The expression of this gene was down regulated by androgens in androgen sensitive prostate cells, but not in the hormone resistant cell lines. The PAR mRNA was detected in all 29 normal human tissues studied and overexpressed in most (67%) of their malignant counterparts. The PAR expression was higher in MCF7 and T47D breast cancer cell lines, as well as in all primary breast tumors studied compared to their normal tissue counterparts. The biological function of this gene is still unknown, but its ubiquitous expression in normal tissues and its overexpression in some malignancies suggest the PAR involvement in certain basic cellular processes and possibly, in malignant transformation.

Retroviral insertional activation of the EVI1 oncogene does not prevent G-CSF-induced maturation of the murine pluripotent myeloid cell line 32Dcl3.

Evi1 is a myeloid-specific protooncogene that encodes 145 kDa and 88 kDa proteins via alternative splicing. Overexpression of the gene via retroviral insertion in murine tumors or chromosomal rearrangement in human tumors is associated with myeloid leukemias and myelodysplasias; however, the mechanism by which such overexpression leads to transformation is not clear. It has been postulated that overexpression of evi1 acts to block normal myelopoiesis. In attempts to assess the effect of overexpression of evi1 on myelopoiesis, we chose to utilize the IL-3-dependent murine 32Dcl3 cell line, which has been shown to differentiate in culture in response to G-CSF. Previous experiments with this cell line, which we have confirmed, showed that overexpression of evi1, mediated by retroviral vector transfer, caused a block to G-CSF-induced cell survival and differentiation. We report here that the naive 32Dcl3 cell line contains a rearrangement of the evi1 locus and constitutively overexpresses evi1 mRNA and protein; this expression is downregulated only slightly during G-CSF-induced myeloid maturation. The steady state levels, molecular weight and DNA binding characteristics of the EVI1 protein in these cells is comparable to that seen in NFS 58, a myeloid leukemia cell line with retroviral insertion at evi1. The observed ability of the murine 32Dcl3 cells to fully differentiate in the presence of G-CSF while evi1 continues to be expressed indicates that, at the levels expressed in naive 32Dcl3, evi1 does not block G-CSF-induced survival and differentiation. Thus, retroviral insertions at evi1 may have been selected for in 32Dcl3 cells due to effects other than that on G-CSF-induced cell survival.

Molecular analysis of Evi1, a zinc finger oncogene involved in myeloid leukemia.

Through chromosomal rearrangements and/or proviral insertions, a number of genes encoding nuclear transcription factors have been identified that play key roles in leukemogenesis. One of these is Evi1, which plays a role in both murine and human myeloid leukemia. The exact mechanism by which Evi1 exerts its leukemogenic effect is not clear, but it may involve the inhibition of terminal differentiation, through the abnormal repression of genes necessary for cellular maturation. Our analysis of the DNA binding characteristics of EVI1 indicate a high degree of specificity, which likely indicates that the protein acts on a tightly defined number of targets in the cell. We are beginning to characterize candidate target genes located in the mouse genome near EVI1 binding sites with the expectation that these will yield insight into EVI1 function both in normal cells and in leukemogenesis.