Mutations in the hydrophobic core of ubiquitin differentially affect its recognition by receptor proteins

Ubiquitin (Ub) is one of the most highly conserved signaling proteins in eukaryotes. In carrying out its myriad functions, Ub conjugated to substrate proteins interacts with dozens of receptor proteins that link the Ub signal to various biological outcomes. Here we report mutations in conserved residues of Ub's hydrophobic core that have surprisingly potent and specific effects on molecular recognition. Mutant Ubs bind tightly to the Ub-associated domain of the receptor proteins Rad23 and hHR23A but fail to bind the Ub-interacting motif present in the receptors Rpn10 and S5a. Moreover, chains assembled on target substrates with mutant Ubs are unable to support substrate degradation by the proteasome in vitro or sustain viability of yeast cells. The mutations have relatively little effect on Ub's overall structure but reduce its rigidity and cause a slight displacement of the C-terminal β-sheet, thereby compromising association with Ub-interacting motif but not with Ub-associated domains. These studies emphasize an unexpected role for Ub's core in molecular recognition and suggest that the diversity of protein-protein interactions in which Ub engages placed enormous constraints on its evolvability.     

Akt phosphorylates and suppresses the transactivation of retinoic acid receptor alpha

The transactivation of nuclear receptors is regulated by both ligand binding and phosphorylation. We previously showed that RARalpha (retinoic acid receptor alpha) phosphorylation by c-Jun N-terminal kinase contributes to retinoid resistance in a subset of NSCLC cells (non-small cell lung cancer cells), but the aetiology of this resistance in the remainder has not been fully elucidated [Srinivas, Juroske, Kalyankrishna, Cody, Price, Xu, Narayanan, Weigel and Kurie (2005) Mol. Cell. Biol. 25, 1054-1069]. In the present study, we report that Akt, which is constitutively activated in NSCLC cells, phosphorylates RARalpha and inhibits its transactivation. Biochemical and functional analyses showed that Akt interacts with RARalpha and phosphorylates the Ser96 residue of its DNA-binding domain. Mutation of Ser96 to alanine abrogated the suppressive effect of Akt. Overexpression of a dominant-negative form of Akt in an NSCLC cell line decreased RAR phosphorylation, increased RAR transactivation and enhanced the growth-inhibitory effects of an RAR ligand. The findings presented here show that Akt inhibits RAR transactivation and contributes to retinoid resistance in a subset of NSCLC cells.     

Rb family proteins differentially regulate distinct cell lineages during epithelial development

pRb, p107 and p130 are important regulators of cell cycle and have extensive overlapping functions; however, only Rb has been shown to be a bone fide tumor suppressor. Defining the overlapping versus distinct pocket protein functions is therefore an important step to understanding the unique role of Rb. Using lung as a model, the present studies demonstrate that pocket proteins are important not only in regulating cell cycle and survival but also in cell lineage specification. An inducible lung-specific Rb knockout strategy was used to demonstrate that Rb is specifically required for restricting neuroendocrine cell fate despite functional compensation for Rb deficiency in other cell types. Ablation of total Rb family function resulted in opposing effects in specification along distinct cell lineages, providing evidence that pocket proteins inhibit neuroendocrine cell fate while being required for differentiation in other cell types. These findings identify a novel role for pocket proteins in cell fate determination, and establish a unique cell lineage-specific function for Rb that explains, at least in part, why Rb and p16 are inactivated in phenotypically distinct carcinomas.     

Analysis of the ligand-binding domain of human retinoic acid receptor alpha by site-directed mutagenesis.

Three subtypes of retinoic acid receptors (RAR), termed RAR alpha, RAR beta, and RAR gamma, have been described. They are composed of different structural domains, including distinct domains for DNA and ligand binding. RARs specifically bind all-trans-retinoic acid (RA), 9-cis-RA, and retinoid analogs. In this study, we examined the functional role of cysteine and arginine residues in the ligand-binding domain of hRAR alpha (hRAR alpha-LBD, amino acids 154 to 462). All conserved cysteine and arginine residues in this domain were mutated by site-directed mutagenesis, and the mutant proteins were characterized by blocking reactions, ligand-binding experiments, transactivation assays, and protease mapping. Changes of any cysteine residue of the hRAR alpha-LBD had no significant influence on the binding of all-trans RA or 9-cis RA. Interestingly, residue C-235 is specifically important in antagonist binding. With respect to arginine residues, only the two single mutations of R-276 and R-394 to alanine showed a dramatic decrease of agonist and antagonist binding whereas the R272A mutation showed only a slight effect. For all other arginine mutations, no differences in affinity were detectable. The two mutations R217A and R294A caused an increased binding efficiency for antagonists but no change in agonist binding. From these results, we can conclude that electrostatic interactions of retinoids with the RAR alpha-LBD play a significant role in ligand binding. In addition, antagonists show distinctly different requirements for efficient binding, which may contribute to their interference in the ligand-inducible transactivation function of RAR alpha.     

In vitro binding of retinoids to the nuclear retinoic acid receptor alpha

We describe a rapid method for measuring in vitro binding properties of new synthetic retinoids to the recently identified nuclear receptor RAR alpha. Transfection of cos-7 cells with the expression vector RAR alpha O produces a 100-fold increase in intracellular RAR alpha concentration which allows us to perform accurate determination of binding parameters of various retinoids. Cytosol and nuclear extracts obtained after freeze drying of the transfected cells are incubated with a new stable tritiated analog of retinoic acid, [3H]CD367. Complete separation between RAR alpha and endogenous cellular retinoic acid binding protein is achieved by high-performance size-exclusion chromatography. These improved techniques provide a useful method for determining binding affinities of analogs to RAR alpha.     

Modulation of retinoic acid receptor alpha activity by lysine methylation in the DNA binding domain

Metabolic labeling and detection with a methylated lysine-specific antibody confirm lysine methylation of RAR alpha in mammalian cells. We previously reported Lys (347) trimethylation of mouse retinoic acid receptor alpha (RAR alpha) in the ligand binding domain (LBD) that affected ligand sensitivity of the dissected LBD. Here we report two monomethylated residues, Lys (109) and Lys (171) identified by LC-ESI-MS/MS in the DNA binding domain (DBD) and the hinge region, which affect retinoic acid (RA) sensitivity, coregulator interaction and heterodimerization with retinoid X receptor (RXR) in the context of the full-length protein. Constitutive negative mutation at Lys (109), but not Lys (171), reduces RA-dependent activation. Methylation at Lys (109) plays a more dominant role than trimethylation at Lys (347) in terms of RA activation of the full-length receptor. Lys (109) is located in a homologous sequence (CEGC K GFFRRS) of the DBD in RARs and is conserved in the nuclear receptor superfamily even across the species boundary. This study uncovers a potential role for monomethylation at Lys (109) in coordinating the synergy between DBD and LBD for ligand-dependent activation of RAR alpha.     

Specific CD45 isoforms differentially regulate T cell receptor signaling.

Multiple isoforms of T cell CD45 tyrosine phosphatase are expressed as a result of alternative RNA splicing among extracellular exons. To discern the presence and identity of distinct functions among CD45 isoforms, we compared thymic T cell activation responses by elevating expression of two CD45 isoforms normally found on quiescent T cells. We report that CD45RABC significantly increased CD4+ thymic T cell proliferation in both a mixed lymphocyte reaction and following anti-T cell receptor (TCR) antibody stimulation. Additionally, CD45RABC enhanced Ca2+ mobilization and phosphotyrosine accumulation, and suppressed the inhibitory effect of anti-CD4 antibodies. By contrast, CD45R0 did not enhance TCR signaling or phosphotyrosine levels in CD4+ thymic T cells and required a TCR co-stimulus to augment cellular proliferation. These studies provide genetic evidence that alternative CD45 isoforms are functionally distinct and disclose a unique mechanism by which T cell immunologic responsiveness can be modified.