PAR (partitioning-defective) proteins, which were first identified in the nematode Caenorhabditis elegans, are essential for asymmetric cell division and polarized growth, whereas Cdc42 mediates establishment of cell polarity. Here we describe an unexpected link between these two systems. We have identified a family of mammalian Par6 proteins that are similar to the C. elegans PDZ-domain protein PAR-6. Par6 forms a complex with Cdc42-GTP, with a human homologue of the multi-PDZ protein PAR-3 and with the regulatory domains of atypical protein kinase C (PKC) proteins. This assembly is implicated in the formation of normal tight junctions at epithelial cell-cell contacts. Thus, Par6 is a key adaptor that links Cdc42 and atypical PKCs to Par3.

The calcium-sensing receptor (CaR) is the major sensor and regulator of extracellular Ca 2+ , whose activity is allosterically regulated by amino acids and pH. Recently, CaR has been identified in the stomach and intestinal tract, where it has been proposed to function in a non-Ca 2+ homeostatic capacity. Luminal nutrients, such as Ca 2+ and amino acids, have been recognized for decades as potent stimulants for gastrin and acid secretion, although the molecular basis for their recognition remains unknown. The expression of CaR on gastrin-secreting G cells in the stomach and their shared activation by Ca 2+ , amino acids, and elevated pH suggest that CaR may function as the elusive physiologic sensor regulating gastrin and acid secretion. The genetic and pharmacologic studies presented here comparing CaR-null mice and wild-type littermates support this hypothesis. Gavage of Ca 2+ , peptone, phenylalanine, Hepes buffer (pH 7.4), and CaR-specific calcimimetic, cinacalcet, stimulated gastrin and acid secretion, whereas the calcilytic, NPS 2143, inhibited secretion only in the wild-type mouse. Consistent with known growth and developmental functions of CaR, G-cell number was progressively reduced between 30 and 90 d of age by more than 65% in CaR-null mice. These studies of nutrient-regulated G-cell gastrin secretion and growth provide definitive evidence that CaR functions as a physiologically relevant multimodal sensor. Medicinals targeting diseases of Ca 2+ homeostasis should be reviewed for effects outside traditional Ca 2+ -regulating tissues in view of the broader distribution and function of CaR.

Multiple endocrine neoplasia type I (MEN1) is an inherited tumor syndrome characterized by development of tumors in multiple endocrine organs. The gene mutated in MEN1 patients, Men1, encodes a nuclear protein, menin. Menin interacts with several transcription factors and inhibits their activities. However, it is unclear whether menin is essential for the repression of the expression of endogenous genes. Here, using menin-null cells, we show that menin is essential for repression of the endogenous IGFBP-2, a gene that can regulate cell proliferation. Additionally, complementation of menin-null cells with wild-type menin, but not with a MEN1 disease-related point mutant, restores the function of menin in repressing IGFBP-2. Consistent with this, the promoter of IGFBP-2 is repressed by wild-type menin, but not by a MEN1-related point mutant. Menin also alters the structure of the chromatin surrounding the promoter of the IGFBP-2 gene, as demonstrated by the deoxyribonuclease I hypersensitivity assay. Furthermore, nuclear localization signals in menin are crucial for repressing the expression of IGFBP-2. Together, these results suggest that menin regulates the expression of the endogenous IGFBP-2 gene at least in part through the promoter of IGFBP-2.

Multiple endocrine neoplasia type I (MEN1), a hereditary tumor syndrome, is characterized by the development of tumors in multiple endocrine organs. The gene mutated in MEN1 patients, Men1, encodes a tumor suppressor, menin. Overexpression of menin leads to inhibition of Rastransformed cells. However, it is unclear whether menin is essential for repression of cell proliferation, and if it is, how it inhibits cell proliferation. Here, we show that targeted disruption of the Men1 gene leads to enhanced cell proliferation, whereas complementation of menin-null cells with menin reduces cell proliferation. Moreover, menin interacts with activator of S-phase kinase (ASK), a component of the Cdc7/ASK kinase complex that is crucial for cell proliferation, but does not appear to alter Cdc7 kinase activity in in vitro kinase assays. We identify the COOH terminus of menin as the domain that mediates the specific interaction with ASK. Notably, wild-type menin completely represses ASK-induced cell proliferation, although it does not obviously affect the steady-state cell cycle profile of ASK-infected cells. Interestingly, disease-related COOHterminal menin mutants that do not interact with ASK completely fail to repress ASK-induced cell proliferation. Together, these findings demonstrate a functional link between menin and ASK in the regulation of cell proliferation.

Ran GTPase is required for nucleocytoplasmic transport of many types of cargo. Several proteins that recognize Ran in its GTP-bound state (Ran⅐GTP) possess a conserved Ran-binding domain (RanBD). Ran-binding protein-1 (RanBP1) has a single RanBD and is required for RanGAP-mediated GTP hydrolysis and release of Ran from nuclear transport receptors (karyopherins). In budding yeast (Saccharomyces cerevisiae), RanBP1 is encoded by the essential YRB1 gene; expression of mouse RanBP1 cDNA rescues the lethality of Yrb1-deficient cells. We generated libraries of mouse RanBP1 mutants and examined 11 mutants in vitro and for their ability to complement a temperature-sensitive yrb1 mutant (yrb1-51 ts ) in vivo. In 9 of the mutants, the alteration was a change in a residue (or 2 residues) that is conserved in all known RanBDs. However, 4 of these 9 mutants displayed biochemical properties indistinguishable from that of wild-type RanBP1. These mutants bound to Ran⅐GTP, stimulated RanGAP, inhibited the exchange activity of RCC1, and rescued growth of the yrb1-51 ts yeast cells. Two of the 9 mutants altered in residues thought to be essential for interaction with Ran were unable to rescue growth of the yrb1 ts mutant and did not bind detectably to Ran in vitro. However, one of these 2 mutants (and 2 others that were crippled in other RanBP1 functions) retained some ability to coactivate RanGAP. A truncated form of RanBP1 (lacking its nuclear export signal) was able to complement the yrb1 ts mutation. When driven from the YRB1 promoter, 4 of the 5 mutants most impaired for Ran binding were unable to rescue growth of the yrb1 ts cells; remarkably, these mutants could nevertheless form ternary complexes with importin-5 or importin-␤ and Ran-GTP. The same mutants stimulated only inefficiently RanGAP-mediated GTP hydrolysis of the Ran⅐GTP⅐importin-5 complex. Thus, the essential biological activity of RanBP1 in budding yeast correlates not with Ran⅐GTP binding per se or with the ability to form ternary complexes with karyopherins, but with the capacity to potentiate Ran-GAP activity toward GTP-bound Ran in these complexes.

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