ZAP is a novel protein phosphatase 1 regulatory subunit localized to the nucleus

Abstract

Nearly all cellular processes are regulated by reversible protein phosphorylation and protein phosphatase 1 (PP 1) is one of the most abundant phosphatases in eukaryotic eel Is. Its intracellular localization, activity and substrate specificity are conferred by a multitude of regulatory subunits, most of which bind via an "RVxF" amino acid motif. ZAP (also ZAP3, ZAPl 13 or YLPMl) possesses an "RVxF" motif and was discovered as a phosphatase­associated protein by microcystin-Sepharose affinity chromatography (Tran et al, (2004) Molecular and Cellular Proteomics, Vol. 3, pp 257). ZAP binds to PPl in a far western overlay, pull-down studies and co-immuno-precipitation and this binding is dependent on ZAP's "RVxF" motif. In addition, ZAP-PPl has no activity against glycogen phosphorylase a unless PP1 is dissociated from the complex by the addition of an "RVxF" containing peptide. Two splice variants of ZAP are present in mammals with apparent molecular weights of 180kDa and 240kDa. Bioinformatic investigation showed that ZAP is conserved in higher eukaryotes, but is missing from fungi, Caenorhabditis elegans and prokaryotes. Its highly conserved carboxy-terminus has similarity to the P-loop kinase domain. Analysis of secondary structure prediction and conserved amino acids suggests that ZAP could have poly-nucleotide kinase activity, however, no activity has been found so far. ZAP is retained specifically on poly-adenylic acid agarose and is di-methylated on arginine residues. Immuno-cytochemistry demonstrated that ZAP is localized in nuclear foci, but does not co­localize with splicing speckles. After transcriptional inhibition by actinomycin D, ZAP re­localized to nucleolar caps. Co-immuno-precipitation with a ZAP antibody revealed several ZAP binding proteins which were identified by peptide mass fingerprinting and western blotting. Heterogeneous nuclear ribonucleoprotein-G (hnRNP-G) is a di-methylated RNA binding protein that also binds to Sam68. The RNA-binding and scaffolding protein Sam68 (src associated in mitosis, 68kDa) associates with tyrosine kinases of the src family and is a binding partner of ZAP. Like most hnRNPs, the association of ZAP, Sam68 and hnRNP-G is dependent on RNA or DNA. The NFl 10/NF45 dimer binds to ZAP as well as to RNA. Lastly, CIA is a nuclear receptor co-regulator and associates with ZAP's amino-terminus. ZAP together with Sam68 and hnRNP-G could form part of a larger hnRNP complex that is involved in mRNA processing. In addition, many ZAP binding proteins are phosphorylated and could be dephosphorylated by ZAP associated PP 1.