05-657 Sigma-AldrichAnti-PP2A Antibody, A subunit, clone 4G7

The Hedgehog (Hh) pathway is evolutionarily conserved and plays critical roles during embryonic development and adult tissue homeostasis. Defective Hh signaling has been linked to a wide range of birth defects and cancers. Hh family proteins regulate the expression of their downstream target genes through the control of proteolytic processing and the transcriptional activation function of Gli transcription factors. Although Hh-dependent regulation of Gli has been studied extensively, other Gli regulatory mechanisms remain relatively unappreciated. Here we report our identification of a novel signaling cascade that controls the stability of Gli proteins. This cascade consists of Daz interacting protein 1 (Dzip1), casein kinase 2 (CK2), and B56 containing protein phosphatase 2As (PP2As). We provide evidence that Dzip1 is involved in a novel Gli turnover pathway. We show that CK2 directly phosphorylates Dzip1 at four serine residues, Ser-664/665/706/714. B56-containing PP2As, through binding to a domain located between amino acid residue 474 and 550 of Dzip1, dephosphorylate Dzip1 on these CK2 sites. Our mutagenesis analysis further demonstrates that the unphosphorylatable form of Dzip1 is more potent in promoting Gli turnover. Consistently, we found that the stability of Gli proteins was decreased upon CK2 inhibition and increased by inhibition of B56-containing PP2As. Thus, reversible phosphorylation of Dzip1, which is controlled by the antagonistic action of CK2 and B56-containing PP2As, has an important impact on the stability of Gli transcription factors and Hh signaling.

Protein phosphatase 2A (PP2A) is one of the most abundantly expressed serine/threonine protein phosphatases. A large body of evidence suggests that PP2A is a tumor suppressor and plays critical roles in regulating apoptosis. PP2A is a heterotrimeric protein complex. Its substrate specificity, localization, and activity are regulated by regulatory subunits of PP2A. A recent study has demonstrated that single nucleotide polymorphism in B56? (PPP2R5E), a B56 family regulatory subunit of PP2A, is associated with human soft tissue sarcoma. This raises the possibility that B56? is involved in tumorigenesis and plays important roles in regulating apoptosis. However, this hypothesis has not been tested experimentally. Our previous studies revealed that B56? regulates a number of developmental signaling pathways during early embryonic patterning. Here we report novel functions of B56? in regulating apoptosis. We provide evidence that B56? has both anti- and pro-apoptotic functions. B56? suppresses p53-independent apoptosis during neural development, but triggers p53-dependent apoptosis. Mechanistically, B56? regulates the p53-dependent apoptotic pathway solely through controlling the stability of p53 protein. In addition to its function in regulating apoptosis, we show that B56? undergoes proteolytic cleavage. The cleavage of B56? is mediated by caspase-3 and occurs on the carboxyl side of an evolutionarily conserved N-terminal DKXD motif. These results demonstrate that B56?, a substrate of caspase-3, is an essential regulator of apoptosis. So far, we have identified an alternative translation isoform and a caspase cleavage product of B56?. The significance of post-transcriptional regulation of B56? is discussed.

Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including development, neuronal signaling, cell cycle regulation, and viral transformation. PP2A has been implicated in Ca(2+)-dependent signaling pathways, but how PP2A is targeted to these pathways is not understood. We have identified two calmodulin (CaM)-binding proteins that form stable complexes with the PP2A A/C heterodimer and may represent a novel family of PP2A B-type subunits. These two proteins, striatin and S/G(2) nuclear autoantigen (SG2NA), are highly related WD40 repeat proteins of previously unknown function and distinct subcellular localizations. Striatin has been reported to associate with the post-synaptic densities of neurons, whereas SG2NA has been reported to be a nuclear protein expressed primarily during the S and G(2) phases of the cell cycle. We show that SG2NA, like striatin, binds to CaM in a Ca(2+)-dependent manner. In addition to CaM and PP2A, several unidentified proteins stably associate with the striatin-PP2A and SG2NA-PP2A complexes. Thus, one mechanism of targeting and organizing PP2A with components of Ca(2+)-dependent signaling pathways may be through the molecular scaffolding proteins striatin and SG2NA.

Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxyl terminus by specific methyltransferase and methylesterase enzymes which have been purified, but not cloned. Carboxymethylation affects PP2A activity and varies during the cell cycle. Here, we report that substitution of glutamine for either of two putative active site histidines in the PP2A C subunit results in inactivation of PP2A and formation of stable complexes between PP2A and several cellular proteins. One of these cellular proteins, herein named protein phosphatase methylesterase-1 (PME-1), was purified and microsequenced, and its cDNA was cloned. PME-1 is conserved from yeast to human and contains a motif found in lipases having a catalytic triad-activated serine as their active site nucleophile. Bacterially expressed PME-1 demethylated PP2A C subunit in vitro, and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. To our knowledge, PME-1 represents the first mammalian protein methylesterase to be cloned. Several lines of evidence indicate that, although there appears to be a role for C subunit carboxyl-terminal amino acids in PME-1 binding, amino acids other than those at the extreme carboxyl terminus of the C subunit also play an important role in PME-1 binding to a catalytically inactive mutant.