Abstract
We describe a serine phosphorylation of the putative metastasis suppressor protein Nm23, and present evidence of its relevance to the signal transduction and tumor metastatic processes. Nm23 was previously demonstrated to exhibit nucleoside diphosphate kinase (NDPK) activity, which transfers a phosphate among nucleoside tri- and diphosphates via an Nm23-phospho-histidine intermediate. Recent data have dissociated the NDPK activity of Nm23 from its phenotypic effects; therefore we have asked whether Nm23 possesses additional biochemical functions. An acid-stable (nonhistidine) phosphorylation was identified on autophosphorylated purified recombinant Nm23 proteins and [32P]orthophosphate-labeled human breast carcinoma and murine melanoma Nm23. Phosphoamino acid analysis identified serine as the acid-stable phosphorylation and serine 44 as the major site of phosphorylation. The acid stable phosphorylation (serine) of Nm23 was inhibited by cAMP in vitro and forskolin in vivo, suggesting that this phosphorylation pathway is regulated in signal transduction. No effect of cAMP was observed on Nm23 NDPK activity. Once phosphorylated, Nm23-phosphoserine can release free phosphate in vitro. The biological relevance of the novel phosphorylation identified herein is suggested by the direct correlation of in vivo Nm23 acid-stable phosphorylation levels, but not Nm23 NDPK activity, with suppression of tumor metastatic potential among control and nm23-1 transfected murine melanoma cells.
Highlights
A Serine Phosphorylationof Nm23, and Not Its Nucleoside Diphosphate Kinase Activity, Correlates with Suppressioonf Tumor Metastatic Potential*
Nm23 protein levels were increased onstratedtoexhibitnucleosidediphosphatekinase with the functional differentiation of the murine heart, brain, (NDPK) activity, which transfers a phosphate among nervous system, and most epithelial tissues in embryogenesis nucleoside tri- and diphosphates via an Nm23-phosph(o6-)
nucleoside diphosphate kinase (NDPK) activity ofNm23from its phenotypiceffects; homologue, abnormal wing discs .Mutation or reduced we have asked whether Nm23 possesses addi-awd expression resulted in normal development during early tional biochemical functions
Summary
Autophosphorylation reactions of HPLC-purified rNm23 proteins were performed fo1r 0min at 37 "C,in TMD buffer containing 10pCi of [Y-~~PIATP. HPLC purified rNm23-H1 (25 pg) was autophosphorylated with [Y-~~PIA(T10P pCi, 7000 Ci/mmol)in TMD buffer for 10 min a t 37 "C. phorylation in 0.3 M trichloroacetic acid may represent btohteh contribution of the phosphohistidine and general protein degradation under these harsh conditions. Panel C , a duplicate sample of [32Plorthophosphatesubjected to the 0.3 M trichloroacetic acid test of acid stability labeled immunoprecipitatedNm23 protein from the experiment shown used previously for rNm23.Incubation of immunoprecipitated in Panel B was incubated in 0.M3 trichloroaceticacid at 100 "C for 75 s in vivo labeled Nm23 in 0.3 M trichloroacetic acid, pH 1.7, at 100 "C for 1 min 15 s prior to electrophoresis resulted in a prior to two-dimensional gel electrophoresis.
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