MAN1 is a vertebrate nuclear inner membrane protein that inhibits Smad signaling downstream of transforming growth factor ␤. MAN1 has an exposed LEM domaincontaining N-terminal region ("MAN1-N"), two transmembrane domains, and an exposed C-terminal domain ("MAN1-C"). Many regions of human MAN1 are homologous to emerin, a LEM domain nuclear protein, loss of which causes Emery-Dreifuss muscular dystrophy (EDMD). To test the hypothesis that MAN1 function might overlap with emerin, we tested different polypeptide fragments of MAN1 for binding to selected partners of emerin. Our findings support this hypothesis. Blot overlay assays and co-immunoprecipitation studies showed that MAN1-C binds the transcription regulators GCL, Btf, and barrier-to-autointegration factor (BAF). BAF binding to this region, which has no LEM domain, was notable. Sequence alignments identified a potential BAF-binding motif, characterized by the conserved residues Ser-Arg-Val, in MAN1-C and two other BAF-binding proteins. The other region, MAN1-N, bound directly to BAF, lamin A, and lamin B1, supporting functional overlap with emerin. Unexpectedly, three independent assays showed that MAN1-N also bound directly to emerin. Proposed MAN1-emerin complexes are discussed in the context of EDMD disease mechanisms and potential in vivo functions.

Oxygen homeostasis represents an essential organizing principle of metazoan evolution and biology. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of transcriptional responses to changes in O 2 concentration. HIF-1 is a heterodimer of HIF-1␣ and HIF-1␤ subunits. O 2 -dependent degradation of the HIF-1␣ subunit is mediated by prolyl hydroxylase, von HippelLindau protein (VHL)/Elongin-C E3 ubiquitin ligase, and the proteasome. O 2 -independent degradation of HIF-1␣ is regulated by the competition of RACK1 and HSP90 for binding to HIF-1␣. RACK1 binding results in the recruitment of the Elongin-C E3 ubiquitin ligase, leading to VHL-independent ubiquitination and degradation of HIF-1␣. In this report, we show that calcineurin inhibits the ubiquitination and proteasomal degradation of HIF-1␣. Calcineurin is a serine/threonine phosphatase that is activated by calcium and calmodulin. The phosphatase activity of calcineurin is required for its regulation of HIF-1␣. RACK1 binds to the catalytic domain of calcineurin and is required for HIF-1␣ degradation induced by the calcineurin inhibitor cyclosporine A. Elongin-C and HIF-1␣ each bind to RACK1 and dimerization of RACK1 is required to recruit Elongin-C to HIF-1␣. Phosphorylation of RACK1 promotes its dimerization and dephosphorylation by calcineurin inhibits dimerization. Serine 146 within the dimerization domain is phosphorylated and mutation of serine 146 impairs RACK1 dimerization and HIF-1␣ degradation. These results indicate that intracellular calcium levels can regulate HIF-1␣ expression by modulating calcineurin activity and RACK1 dimerization.All metazoan organisms possess physiological systems to regulate the delivery and/or utilization of O 2 . Hypoxia-inducible factor 1 (HIF-1) 3 is a critical mediator of adaptive responses to reduced oxygen availability in many developmental, physiological, and pathological contexts through its transcriptional regulation of genes that encode proteins required for tissue oxygen delivery and energy metabolism (1-4). HIF-1 is required for embryonic vascularization. Mouse embryos with complete HIF-1␣ deficiency or expression of a dominant negative form of HIF-1 in endothelial cells fail to develop proper vascularization and die at embryonic days 10 -11 (4 -6). HIF-1-dependent induction of angiogenic factors promotes vascularization and cardiac function in the initial compensated phase of left ventricular hypertrophy caused by pressure overload (7). In contrast to this adaptive response, HIF-1 contributes to the pathogenesis of hypoxia-induced pulmonary hypertension and right ventricular hypertrophy (8). HIF-1 also plays an important pathogenic role in many critical aspects of cancer biology, including cell immortalization, energy metabolism, vascularization, invasion, metastasis, and resistance to radiation and chemotherapy (9). HIF-1 is a heterodimeric transcription factor composed of a HIF-1␤ subunit, which is constitutively expressed, and a HIF-1␣ subunit, the expression and transcriptional activity of which are regul...

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