Protein phosphatase 2A is a heterotrimeric protein serine/threonine phosphatase consisting of a 36-kDa catalytic C subunit, a 65-kDa structural A subunit, and a variable regulatory B subunit. The B subunits determine the substrate specificity of the enzyme. There have been three families of cellular B subunits identified to date: B55, B56 (B'), and PR72/130. We have now cloned five genes encoding human B56 isoforms. Polypeptides encoded by all but one splice variant (B56gamma1) are phosphoproteins, as shown by mobility shift after treatment with alkaline phosphatase and metabolic labeling with [32P]phosphate. All labeled isoforms contain solely phosphoserine. Indirect immunofluorescence microscopy demonstrates distinct patterns of intracellular targeting by different B56 isoforms. Specifically, B56alpha, B56beta, and B56epsilon complexed with the protein phosphatase 2A A and C subunits localize to the cytoplasm, whereas B56delta, B56gamma1, and B56gamma3 are concentrated in the nucleus. Two isoforms (B56beta and B56delta) are highly expressed in adult brain; here we show that mRNA for these isoforms increases severalfold when neuroblastoma cell lines are induced to differentiate by retinoic acid treatment. These studies demonstrate an increasing diversity of regulatory mechanisms to control the activity of this key intracellular protein phosphatase and suggest distinct functions for isoforms targeted to different intracellular locations.

CD4(+) T helper cells differentiate into T helper 1 (Th1) or Th2 effector lineages, which orchestrate immunity to different types of microbes. Both Th1 and Th2 differentiation can be induced by Notch, but what dictates which of these programs is activated in response to Notch is not known. By using T cell-specific gene ablation of the Notch effector RBP-J or the Notch1 and 2 receptors, we showed here that Notch was required on CD4(+) T cells for physiological Th2 responses to parasite antigens. GATA-3 was necessary for Notch-induced Th2 differentiation, and we identified an upstream Gata3 promoter as a direct target for Notch signaling. Moreover, absence of GATA-3 turned Notch from a Th2 inducer into a powerful inducer of Th1 differentiation. Therefore, Gata3 is a critical element determining inductive Th2 differentiation and limiting Th1 differentiation by Notch.

Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism. The ability of this widely distributed heterotrimeric enzyme to act on a diverse array of substrates is largely controlled by the nature of its regulatory B subunit. Only two gene families encoding endogenous B subunits have been cloned to date, although the existence of several additional regulatory subunits is likely. We have identified by two-hybrid interaction a new human gene family encoding PP2A B subunits. This family, denoted B56, contains three distinct genes, one of which is differentially spliced. B56 polypeptides co-immunoprecipitate with PP2A A and C subunits and with an okadaic acid-inhibitable, heparinstimulated phosphatase activity. The three B56 family members are 70% identical to each other but share no obvious homology with previously identified B subunits. These phosphatase regulators are differentially expressed, with B56␣ and B56␥ highly expressed in heart and skeletal muscle and B56␤ highly expressed in brain. The identification of this novel phosphatase regulator gene family will facilitate future studies on the control of protein dephosphorylation and the role of PP2A in cellular function. Protein phosphatase 2A (PP2A)1 is a major intracellular phosphatase that regulates such diverse cellular processes as DNA replication, transcription, signal transduction, and intermediary metabolism (1-3). PP2A is a heterotrimer, containing A, B, and C subunits. The catalytic activity of PP2A resides in the C subunit, a 36-kDa protein encoded by two 97% identical genes. The C subunit binds stably to the carboxyl-terminal region of the A subunit, a 65-kDa rod-shaped polypeptide consisting of 15 imperfect repeats. The B subunits bind to the amino-terminal region of the A subunit (Fig. 1A) and determine the substrate specificity of the complex (4 -8). Three distinct B subunits have been biochemically isolated from a variety of mammalian tissues (9 -14), and several studies have suggested the existence of additional B subunits (15, 16). Additionally, several DNA tumor viruses encode polypeptides that can function as PP2A B subunits (17-19). The B subunits purified to date migrate in SDS-PAGE with the apparent molecular masses of 54 kDa (B54), 55 kDa (B55), and 72 kDa (B72). Three cDNAs encoding 55-kDa B subunits have been identified (10,11,20); the B55 family members are 80 -90% identical, and their level of expression varies by tissue type. A cDNA encoding the 72-kDa B subunit has also been cloned, and a splice variant encoding a 130-kDa protein has been identified (12). The sequence of the 54-kDa B subunit cDNA has not yet been reported. Interestingly, the amino acid sequences of the B55 and B72 subunits and the viral PP2A binding proteins show little homology to each other; thus, no common motif mediating the interaction of the B subunit with the PP2A A and C subunits has been discovered.Heterotrimeric PP2A enzymes with different B subunits have distinct substrate spe...

BackgroundAlagille syndrome is a developmental disorder caused predominantly by mutations in the Jagged1 (JAG1) gene, which encodes a ligand for Notch family receptors. A characteristic feature of Alagille syndrome is intrahepatic bile duct paucity. We described previously that mice doubly heterozygous for Jag1 and Notch2 mutations are an excellent model for Alagille syndrome. However, our previous study did not establish whether bile duct paucity in Jag1/Notch2 double heterozygous mice resulted from impaired differentiation of bile duct precursor cells, or from defects in bile duct morphogenesis.Methodology/Principal FindingsHere we characterize embryonic biliary tract formation in our previously described Jag1/Notch2 double heterozygous Alagille syndrome model, and describe another mouse model of bile duct paucity resulting from liver-specific deletion of the Notch2 gene.Conclusions/SignificanceOur data support a model in which bile duct paucity in Notch pathway loss of function mutant mice results from defects in bile duct morphogenesis rather than cell fate specification.

We previously determined that Notch2, and not Notch1 was required for forming proximal nephron segments. The dominance of Notch2 may be conserved in humans, since Notch2 mutations occur in Alagille syndrome (ALGS) 2 patients, which includes renal complications. To test whether mutations in Notch1 could increase the severity of renal complications in ALGS, we inactivated conditional Notch1 and Notch2 alleles in mice using a Six2-GFP∷Cre. This BAC transgene is expressed mosaically in renal epithelial progenitors but uniformly in cells exiting the progenitor pool to undergo mesenchymal to epithelial transition. Although delaying Notch2 inactivation had a marginal effect on nephron numbers, it created a sensitized background in which the inactivation of Notch1 severely compromised nephron formation, function and survival. These and additional observations indicate that Notch1 in concert with Notch2 contributes to the morphogenesis of renal vesicles into S-shaped bodies in a RBP-J dependent manner. A significant implication is that elevating Notch1 activity could improve renal functions in ALGS2 patients. As proof of principle, we determined that conditional inactivation of Mint, an inhibitor of Notch-RBP-J interaction, resulted in a moderate rescue of Notch2 null kidneys, implying that temporal blockage of Notch signaling inhibitors downstream of receptor activation may have therapeutic benefits for ALGS patients.