We used the interaction trap, a yeast genetic selection for interacting proteins, to isolate human cyclin-dependent kinase interactor 1 (Cdi1). In yeast, Cdi1 interacts with cyclin-dependent kinases, including human Cdc2, Cdk2, and Cdk3, but not with Ckd4. In HeLa cells, Cdi1 is expressed at the G1 to S transition, and the protein forms stable complexes with Cdk2. Cdi1 bears weak sequence similarity to known tyrosine and dual specificity phosphatases. In vitro, Cdi1 removes phosphate from tyrosine residues in model substrates, but a mutant protein that bears a lesion in the putative active site cysteine does not. Overexpression of wild-type Cdi1 delays progression through the cell cycle in yeast and HeLa cells; delay is dependent on Cdi1 phosphatase activity. These experiments identify Cdi1 as a novel type of protein phosphatase that forms complexes with cyclin-dependent kinases.

The mammalian cilium protrudes from the apical/lumenal surface of polarized cells and acts as a sensor of environmental cues. Numerous developmental disorders and pathological conditions have been shown to arise from defects in cilia-associated signaling proteins. Despite mounting evidence that cilia are essential sites for coordination of cell signaling, little is known about the cellular mechanisms controlling their formation and disassembly. Here, we show that interactions between the prometastatic scaffolding protein HEF1/Cas-L/NEDD9 and the oncogenic Aurora A (AurA) kinase at the basal body of cilia causes phosphorylation and activation of HDAC6, a tubulin deacetylase, promoting ciliary disassembly. We show that this pathway is both necessary and sufficient for ciliary resorption and that it constitutes an unexpected nonmitotic activity of AurA in vertebrates. Moreover, we demonstrate that small molecule inhibitors of AurA and HDAC6 selectively stabilize cilia from regulated resorption cues, suggesting a novel mode of action for these clinical agents.

Since their introduction, the interaction trap and other two-hybrid systems have been used to study protein-protein interactions. Despite their general use, little is known about the extent to which the degree of protein interaction determined by two-hybrid approaches parallels the degree of interaction determined by biochemical techniques. In this study, we used a set of lexAop-LEU2 and lexAop-lacZ reporters to calibrate the interaction trap. For the calibration, we used two sets of proteins, the Myc-Max-Mxi1 helix-loop-helix proteins, and wild-type and dimerization-defective versions of the lambda cI repressor. Our results indicate that the strength of interaction as predicted by the two-hybrid approach generally correlates with that determined in vitro, permitting discrimination of high-, intermediate-, and low-affinity interactions, but there was no single reporter for which the amount of gene expression linearly reflected affinity measured in vitro. However, some reporters showed thresholds and only responded to stronger interactions. Finally, some interactions were subject to directionality, and their apparent strength depended on the reporter used. Taken together, our results provide a cautionary framework for interpreting affinities from two-hybrid experiments.Biological systems depend on interactions between protein components. These interactions affect such diverse processes as the coordination of signal transduction by assembly of multisubunit complexes (57, 58), the regulation of apoptosis by the sequestration of Bax (54), and the control of gene expression through the selective association of transcription factors (19). Efforts to understand the functions of proteins often include identification and characterization of other cellular proteins with which they can interact. While some protein interactions are of high affinity and are easily detectable by physical techniques, a number of biologically important interactions, such as those of many enzymes with their substrates, are often relatively weak or transient and are not easily detectable by these methods.A number of approaches for studying protein association are in use, including cosedimentation through gradients, coimmunoprecipitation of purified proteins, assay of DNA binding activity for proteins that must dimerize to recognize a DNA site, and assay by two-hybrid systems (20) such as the interaction trap (31). In the last approach, a first protein (P1, or ''bait'') is fused to a known DNA-binding domain such as LexA (10) or GAL4 (41) and a second protein (P2) is fused to a transcriptional activation domain (AD). Coexpression of the two chimeric proteins in yeast cells in which the cognate binding site for the DNA-binding domain is located upstream of a reporter gene results in transcriptional activation of the reporter by the P2-fused AD if the chimeric proteins associate.Two-hybrid/interaction trap approaches have gained considerable popularity because they can detect novel interacting proteins that interact with a given bait by substituting...

Interactions of the BcI-2 protein with itself and other members of the Bcl-2 family, including Bcl-X-L, Bcl-X-S, Mci-i, and Bax, were explored with a yeast twohybrid system. Fusion proteins were created by linking BcI-2 family proteins to a LexA DNA-binding domain or a B42 trans-activation domain. Protein-protein interactions were examined by expression of these fusion proteins in Saccharomyces cerevisiae having a lacZ ((-galactosidase) gene under control of a LexA-dependent operator. This approach gave evidence for Bcl-2 protein homodimerization. Bcl-2 also interacted with Bcl-X-L and Mcl-i and with the dominant inhibitors Bax and Bcl-X-S. Bd-X-L displayed the same pattern of combinatorial interactions with Bd-2 family proteins as Bc1-2. Use of deletion mutants of Bc-2 suggested that BcI-2 homodimerization involves interactions between two distinct regions within the Bcl-2 protein, since a LexA protein containing Bcl-2 amino acids 83-218 mediated functional interactions with a B42 fusion protein contaiing Bcl-2 amino acids 1-81 but did not complement a B42 fusion protein containing BcI-2 amino acids 83-218. In contrast to LexA/Bcl-2 fusion proteins, expression of a LexA/Bax protein was lethal to yeast. This cytotoxicity could be abrogated by B42 fusion proteins containing BcI-2, Bcl-X-L, or Mci-i but not those containing Bcl-X-S (an alternatively spliced form of Bcl-X that lacks a well-conserved 63-amino acid region). The findings suggest a model whereby Bax and Bcl-X-S differentially regulate Bcd-2 function, and indicate that requirements for Bcl-2/Bax heterodimerization may be different from those for Bcl-2/Bcl-2 homodimerization.The bcl-2 gene becomes dysregulated in a wide variety of human cancers and contributes to neoplastic cell expansion by prolonging cell survival rather than by accelerating rates of cellular proliferation. Specifically, bcl-2 blocks programmed cell death, a physiological process that normally ensures a homeostatic balance between cell production and cell turnover in most tissues with self-renewal capacity and which often involves characteristic changes in cell morphology termed apoptosis. In fact, Bc1-2 can prevent or delay apoptosis induced by a wide variety of stimuli, including growth factor deprivation, alterations in Ca2+, free radicals, cytotoxic lymphokines, some types of viruses, radiation, and most chemotherapeutic drugs, suggesting that this oncoprotein controls a common final pathway involved in cell death regulation (reviewed in refs. 1 and 2).The mechanism by which Bcl-2 prevents cell death remains enigmatic, as the predicted amino acid sequence of the 26-kDa human Bcl-2 protein (239 aa) has no significant homology with other proteins whose biochemical activity is known. Recently, however, Bcl-2 has been shown to interact with a low molecular weight GTPase member of the Ras family, p23-R-Ras (3), and also can be coimmunoprecipitated with the serine/threonine-specific protein kinase Raf-1 (4). Thus, Bcl-2 may somehow regulate a signal transduction pathway involving...

Temporally and spatially controlled activation of the Aurora-A kinase (AURKA) is regulates centrosome maturation, entry into mitosis, formation and function of the bipolar spindle, and cytokinesis. Genetic amplification, and mRNA and protein overexpression of Aurora-A are common in many types of solid tumor, and associated with aneuploidy, supernumerary centrosomes, defective mitotic spindles, and resistance to apoptosis. These properties have led Aurora-A to be considered a high value target for development of cancer therapeutics, with multiple agents currently in early phase clinical trials. More recently, identification of additional, non-mitotic functions and means of activation of Aurora-A during interphase neurite elongation and ciliary resorption have significantly expanded understanding of its function, and may offer insights into clinical performance of Aurora-A inhibitors. We here review mitotic and non-mitotic functions of Aurora-A, discuss Aurora-A regulation in the context of protein structural information, and evaluate progress in understanding and inhibiting Aurora-A in cancer.