AN ACTH-ACTIVATED TYROSINE PHOSPHATASE IN RAT ADRENAL ZONA FASCICULATA IS PTP-PEST

GOROSTIZAGA A; BRION L; MORI SEQUEIROS GARCÍA M; CORNEJO MACIEL F; PODESTÁ E J; MENDEZ C.F.; PAZ C.

San Francisco, U.S.A.

Congreso; XIII Adrenal Cortex Conference; 2008

We have previously demonstrated that ACTH activates a 115 kDa tyrosine phosphatase (PTP115) in rat adrenal zona fasciculata (ZF). Although we determined its molecular weight by an in-gel PTP assay, the identity of this protein remained elusive. In order to characterize PTP115, we performed its partial purification from rat adrenal ZF. The cytosolic extracts were subjected to ammonium sulphate precipitation, DEAE-cellulose chromatography and filtration through filters with a 100 kDa cut-off.  The analysis of this partially-purified sample by in-gel PTP assay did not show other PTPs. This sample was then subjected to in vitro phosphorylation by PKA and its phosphatase activity determined using a synthetic substrate. Lineweaver-Burk analysis showed that phosphorylation of partially-purified PTP115 increases its Vmax. Western blot analysis using an antibody against PTP-PEST revealed a 115 kDa band in the cytosolic fraction. This antibody also detected a single band with Rf similar to the band with PTP activity in the partial purified samples. In addition, we also identified the mRNA of PTP-PEST in Y1 adrenocortical cells by RT-PCR. Together, our results indicate that the ACTH-activated PTP115 is PTP-PEST, and that PKA phosphorylation increases its enzymatic activity.