SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27.

Degradation of the mammalian cyclin-dependent kinase (CDK) inhibitor p27 is required for the cellular transition from quiescence to the proliferative state. The ubiquitination and subsequent degradation of p27 depend on its phosphorylation by cyclin-CDK complexes. However, the ubiquitin-protein ligase necessary for p27 ubiquitination has not been identified. Here we show that the F-box protein SKP2 specifically recognizes p27 in a phosphorylation-dependent manner that is characteristic of an F-box-protein-substrate interaction. Furthermore, both in vivo and in vitro, SKP2 is a rate-limiting component of the machinery that ubiquitinates and degrades phosphorylated p27. Thus, p27 degradation is subject to dual control by the accumulation of both SKP2 and cyclins following mitogenic stimulation.

Resolution and reconstitution of ion-transport systems.

1. Oxidative phosphorylation was reconstituted with a mitochondrial proton pump (oligomycin-sensitive ATPase) and segments of the oxidation chain (cytochrome oxidase or DPNH-Q1 reductase). A proton pump of bacteriorhodopsin substituted for the respiratory chain components, giving rise to light-induced ATP formation. 2. Since oxidative phosphorylation has thus become a special case of the problem of ion translocation in general, we have investigated and reconsituted other pumps. The reconstituted Ca++ pump of sarcoplasmic reticulum consists of two factors, the Ca++-dependent ATPase and a heat-stable coupling factor. 3. Other information obtained from reconstitution experiments include the role of asymmetry in organized membranes and the specificity of protein-phospholipid interaction. 4. Purified preparations of Ca++-ATPase catalyze the formation of ATP from Pi and ADP in a stepwise reaction stoichiometric with the enzyme and dependent on Ca++.

Speech coding strategies and revised cochlear implant candidacy: an analysis of post-implant performance.

OBJECTIVE: Technological advances in cochlear implant systems on which a sequence of speech coding strategies have been implemented seem to have resulted in improved speech perception. However, changing selection criteria for implantation have coincided with evolving technology and may confound post-implantation speech perception performance. This study compares speech coding strategy with speech perception performance in severe and profound postlingually deafened adults using one of three successive generations of Nucleus Cochlear Implant speech processors (i.e., Mini Speech Processor, Spectra 22, and SPrint) implementing three speech coding strategies (i.e., MPEAK, SPEAK, and Advanced Combination Encoders; Cochlear Corporation, Englewood, CO, U.S.A.). STUDY DESIGN: Four cohorts of patients were retrospectively reviewed. SETTING: Multicenter, tertiary referral cochlear implant programs in Ontario, Canada. METHODS: Four cohorts of patients (n = 139) were identified based on preimplant audiological measures, duration of deafness, device type, and speech coding strategy. Word and sentence recognition scores at 12 months after implantation were compared using MPEAK with SPEAK22 implemented on the Nucleus 22 speech processors (Mini Speech Processor and Spectra22, respectively) and SPEAK24 as well as Advanced Combination Encoders implemented on the Nucleus 24 SPrint processor. RESULTS: Open-set speech recognition batteries revealed significant improvements in word and sentence scores as advancing technology implemented new speech coding strategies. Subgroup analysis of profoundly deafened patients supported this. Analysis of covariance confirmed that the measured differences could not be accounted for by changing selection criteria for implantation. CONCLUSION: Improvements in performance can be attributed to evolving speech coding strategies and speech processors rather than to differences in preimplant candidacy.

Relevance of protease "inhibitor" to the ATP-ubiquitin proteolytic system.

ATP-dependent proteolysis in reticulocyte extracts is stimulated by ubiquitin, a polypeptide which is covalently conjugated to proteins. It has been proposed that ATP and ubiquitin act by repressing an inhibitor of an ATP-independent protease, rather than by conjugation to substrate proteins [Speiser, S. and Etlinger, J.D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3577-3580]. We find that the inhibitor preparation used by these authors contains a positively required factor of the ATP-ubiquitin proteolytic system, which can be separated from two types of protease inhibitors by gel filtration chromatography. The following observations indicate that the "inhibitors" are endogenous protease substrates which compete with the labeled substrate: (a) inhibition is competitive with exogenous substrate; (b) inhibition is abolished by a preincubation of "inhibitor" with protease prior to the addition of labeled substrate. These findings are not consistent with the notion that the inhibitors play a regulatory role in the ATP-ubiquitin proteolytic pathway.

A coupling factor from sarcoplasmic reticulum required for the translocation of Ca2+ ions in a reconstituted Ca2+ATPase pump.

1. During purification of the Ca2+ATPase from sarcoplasmic reticulum of rabbit muscle, different fractions with similar Ca2+ATPase activity were found to vary greatly in their ability to catalyze 45Ca2+ translocation in reconstituted liposomal systems. 2. A heat-stable fraction isolated from the fraction most active in Ca2+ translocation enhanced several-fold the Ca2+ translocation rate of the least active fraction. It also increased the ratio of Ca2+ translocation to ATP hydrolysis over 5-fold. The properties of the coupling factor resemble those of the proteolipid previously described by MacLennan et al. (MACLENNAN, D.H., YIP, C. C., ILES, G. H., and SEAMAN, P. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 469-478). 3. When the heat-stable factor was added to either sarcoplasmic reticulum fragments or to liposomes after, rather than before, reconstitution, it acted as an ionophore abolishing Ca2+ translocation.

Fusion of proteoliposomes and cells. ATP-dependent Ca2+ uptake into erythrocytes catalyzed by Ca2+-ATPase from skeletal muscle.

Purified Ca2+-ATPase from rabbit skeletal muscle has been incorporated into intact erythrocyte membranes by a two-step procedure. The isolated protein was reconstituted into proteoliposomes composed of phosphatidylethanolamine, phosphatidylcholine, and cardiolipin (50:20:30%, respectively). The resulting proteoliposomes were fused with erythrocytes in presence of La3+, Ca2+, or Mg2+. Subsequently, 45Ca uptake into the cells could be demonstrated. It was dependent on externally added ATP, inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate, and enhanced by inactivation of the endogenous Ca2+-ATPase which catalyzes Ca2+ extrusion from the cells. The insertion of the protein did not induce cell lysis, but the cells did become more fragile. Functional insertion of isolated membrane proteins into cell membranes allows a new approach to research of plasma membranes.

A multicomponent system that degrades proteins conjugated to ubiquitin. Resolution of factors and evidence for ATP-dependent complex formation.

It was found previously that proteins conjugated to ubiquitin are degraded by an ATP-dependent enzyme system, but the mode of action of this system was unknown. We have resolved from reticulocyte extracts three factors that are required for the ATP-dependent breakdown of 125I-lysozyme-ubiquitin conjugates. Two of the factors interact with ATP, as shown by their protection against heat inactivation by the nucleotide. When the three factors are incubated with 125I-lysozyme-ubiquitin conjugates and ATP, there is a lag of 4-6 min in the formation of acid-soluble products before the onset of rapid proteolysis. The lag can be abolished by incubation of the three factors with MgATP prior to the addition of the substrate. This "activation" process does not take place if any of the three factors is omitted from preincubation (and added subsequently) or when ATP is replaced by a nonhydrolyzable analog. Analysis of size distribution by glycerol density gradient centrifugation showed that following incubation of the three factors with MgATP, a high molecular mass (greater than 1000 kDa) activity is formed. That the high molecular weight form is a complex of the three factors is indicated by the finding that its formation is accompanied by a corresponding decrease in the levels of the free forms of all three factors. Complex formation seems to be similar to the activation process with regard to time course, requirements for ATP and Mg2+, partial effect of CTP, and lack of effect of nonhydrolyzable ATP analogs. It is suggested that one role of ATP in conjugate breakdown is the formation of an active multienzyme complex.

The protein substrate binding site of the ubiquitin-protein ligase system.

In order to gain insight into the mechanisms that determine the selectivity of the ubiquitin proteolytic pathway, the protein substrate binding site of the ubiquitin-protein ligase system was identified and examined. Previous studies had shown that the ligase system consists of three components: a ubiquitin-activating enzyme (E1), ubiquitin-carrier protein (E2), and a third enzyme, E3, the mode of action of which has not been defined. E3 from rabbit reticulocytes was further purified by a combination of affinity chromatography, hydrophobic chromatography, and gel filtration procedures. A 180-kDa protein was identified as the subunit of E3. Two independent methods indicate that E3 has the protein binding site of the ubiquitin ligase system. These are the chemical cross-linking of 125I-labeled proteins to the E3 subunit and the functional conversion of enzyme-bound labeled proteins to ubiquitin conjugates in pulse-chase experiments. The trapping of E3-bound protein for labeled product formation was allowed by the slow dissociation of E3 X protein complex. The specificity of binding of different proteins to E3, examined by both methods, showed a direct correlation with their susceptibility to degradation by the ubiquitin system. Proteins with free alpha-NH2 groups, which are good substrates, bind better to E3 than corresponding proteins with blocked NH2 termini, which are not substrates. Oxidation of methionine residues to sulfoxide derivatives greatly increases the susceptibility of some proteins to ligation with ubiquitin, with a corresponding increase in their binding to E3. However, a protein derivative which was subjected to both amino group modification and oxidation binds strongly to the enzyme, even though it cannot be ligated to ubiquitin. It thus seems that the substrate binding site of E3 participates in determining the specificity of proteins that enter the ubiquitin pathway of protein degradation.

Phospholipid-protein interactions in the Ca2+-adenosine triphosphatase of sarcoplasmic reticulum.

Ca2+-adenosine triphosphatase from sarcoplasmic reticulum has been delipidated by gel filtration through a Sephadex G-200 column equilibrated with buffer containing cholate. The delipidated Ca2+-adenosine triphosphatase had negligible adenosine triphosphatase activity, but up to 50% of the ATPase activity was restored when the delipidated enzyme was recombined with phosphilipids. It was shown with the delipidated preparation that the phosphorylation of the enzyme by either ATP or Pi was entirely dependent on phospholipids. Among the purified phospholipids, phosphatidylcholine reactivated the adenosine triphosphatase activity better than phosphatidylethanolamine. Vesicles capable of translocating Ca2+ were reconstituted from delipidated Ca2+-adenosine triphosphatase and phosphatidylethanolamine, but not with phosphatidylcholine alone. We conclude that the firmly bound phospholipids which are purified together with the adenosine triphosphatase protein are not essential for the pump since they can be substituted by phosphatidylethanolamine isolated from soybeans.

ATP-dependent incorporation of 20S protease into the 26S complex that degrades proteins conjugated to ubiquitin.

Previous studies have indicated that the ATP-dependent 26S protease complex that degrades proteins conjugated to ubiquitin is formed by the assembly of three factors in an ATP-requiring process. We now identify one of the factors as the 20S "multicatalytic" protease, a complex of low molecular weight subunits widely distributed in eukaryotic cells. Comparison of the subunit compositions of purified 20S and 26S complexes indicates that the former is an integral part of the latter. By the use of detergent treatment to activate latent protease activity, we show that the 20S protease becomes incorporated into the 26S complex in the ATP-dependent assembly process. It thus seems that the 20S protease is the "catalytic core" of the 26S complex of the ubiquitin proteolytic pathway.

Immunochemical analysis of the turnover of ubiquitin-protein conjugates in intact cells. Relationship to the breakdown of abnormal proteins.

Previous studies in a cell-free proteolytic system from reticulocytes indicated that the conjugation of ubiquitin with proteins plays a role in protein breakdown. To examine some of the physiological functions of the ubiquitin conjugation system, and immunochemical method was developed for the isolation of ubiquitin-protein conjugates from intact cells. A specific antiserum was raised against ubiquitin and purified by affinity chromatography on ubiquitin-Sepharose. When cells are labeled with tryptophan (which is missing from ubiquitin), labeled immunoreactive material isolated by the antibody is derived from the protein moiety of ubiquitin-protein conjugates. There is a marked increase in the labeling of ubiquitin-protein conjugates during the formation of abnormal proteins in reticulocytes (induced by the incorporation of amino acid analogs), suggesting that proteins with abnormal structure are more readily conjugated to ubiquitin than most normal proteins. Essentially similar, although less marked, effects of amino acid analogs were observed in Ehrlich ascites cells. When further protein synthesis was blocked with cycloheximide, ubiquitin conjugates decayed more extensively than the corresponding average labeled cellular proteins. This is consistent with the interpretation that a considerable part of ubiquitin conjugates is derived from a pool of rapidly degradable proteins.

Energy-linked nicotinamide-nucleotide transhydrogenase. Light-driven transhydrogenase catalyzed by transhydrogenase from beef heart mitochondria reconstituted with bacteriorhodopsin.

Purified nicotinamide-nucleotide transhydrogenase from beef heart mitochondria was co-reconstituted with bacteriorhodopsin to from transhydrogenase-bacteriorhodopsin vesicles that catalyze a 20-fold light-dependent and uncoupler-sensitive stimulation of the reduction of NADP+ and NADP+ analogs by NADH and a 50-fold shift of the nicotinamide nucleotide ratio. In the presence of light, the transhydrogenase-bacteriorhodopsin vesicles catalyzed a pronounced light intensity-dependent inward proton pumping as indicated by a pH shift of the medium. As indicated by pH shifts, proton pumping by the bacteriorhodopsin essentially paralleled the light-driven transhydrogenase. Addition of valinomycin increased the pH shift twice with a concomitant 50% inhibition of the light-driven transhydrogenase, whereas nigericin inhibited the pH shift completely and the light-driven transhydrogenase partially. Taken together, these results suggest that transhydrogenase and bacteriorhodopsin interact through a delocalized proton-motive force. Possible partial reactions of transhydrogenase were investigated with transhydrogenase-bacteriorhodopsin vesicles energized by light. Reduction of oxidized 3-acetylpyridine adenine dinucleotide by NADH, previously claimed to represent partial reactions, was found to require NADPH. Similarly, reduction of thio-NADP+ by NADPH required NADH. It is concluded that these reactions do not represent partial reactions.

Role of the alpha-amino group of protein in ubiquitin-mediated protein breakdown.

Previous studies suggest that the conjugation of ubiquitin to NH2 groups of proteins is required for protein breakdown. We now show that the selective modification of NH2-terminal alpha-NH2 groups of globin and lysozyme prevents their degradation by the ubiquitin proteolytic system from reticulocytes. The conjugation by ubiquitin of epsilon-NH2 groups of lysine residues, usually seen in multiples, was also inhibited in alpha-NH2-blocked proteins. Naturally occurring N alpha-acetylated proteins are not degraded by the ubiquitin system at a significant rate, while their nonacetylated counterparts from other species are good substrates. This suggests that one function of N alpha-acetylation of cellular proteins is to prevent their degradation by the ubiquitin system. alpha-NH2-blocked proteins can have their activity as substrates for degradation increased by incorporation of alpha-NH2 groups through the introduction of polyalanine side chains. Proteins in which most epsilon-NH2 groups are blocked but the alpha-NH2 group is free are degraded by the ubiquitin system, but at a reduced rate. It is therefore suggested that the exposure of a free NH2 terminus of proteins is required for degradation and probably initiates the formation of ubiquitin conjugates committed for degradation.

Ubiquitin C-terminal hydrolase activity associated with the 26 S protease complex.

In the ubiquitin (Ub) system for protein degradation, proteins ligated to Ub are degraded by an ATP-dependent 26 S protease complex. During or after proteolysis, free Ub is regenerated, but the mechanisms of Ub release remained unknown. It was previously observed that free Ub is released from a Ub-histone conjugate by an ATP-dependent activity present in partially purified preparations of 26 S complex, but the relationship of this activity to protein breakdown was not established. We now show that purified preparations of 26 S complex release free Ub from conjugates that are good substrates for proteolysis, such as conjugates of lysozyme with reductively methylated Ub. The activity that releases free Ub co-migrates with the 26 S protease complex in glycerol density gradient centrifugation, indicating that the responsible Ub C-terminal hydrolase is an integral part of the 26 S complex. Complex-associated hydrolase can also act on adducts in which a single Ub unit is attached to protein, such as a bacterially expressed construct in which the C terminus of Ub is fused to the alpha-NH2 group of a fragment of Ub that contains 60% of its N-terminal region. In all cases, Ub release is insensitive to Ub-aldehyde (an inhibitor of some Ub C-terminal hydrolases) and is stimulated by MgATP. ATP cannot be replaced by beta, gamma-nonhydrolyzable analogs, but it can be substituted by CTP and GTP. The nucleotide specificity of Ub release by the 26 S complex is similar to that observed previously for conjugate proteolysis and nucleotide hydrolysis. It thus seems that the activity of the Ub C-terminal hydrolase associated with the 26 S complex is tightly coupled to the proteolytic action of the complex, and it may have a role in the release of Ub from linkage to amino groups of the protein substrate at the final stages of the Ub proteolytic pathway.

E2-C, a cyclin-selective ubiquitin carrier protein required for the destruction of mitotic cyclins.

Ubiquitin-dependent proteolysis of the mitotic cyclins A and B is required for the completion of mitosis and entry into the next cell cycle. This process is catalyzed by the cyclosome, an approximately 22S particle that contains a cyclin-selective ubiquitin ligase activity, E3-C, that requires a cyclin-selective ubiquitin carrier protein (UBC) E2-C. Here we report the purification and cloning of E2-C from clam oocytes. The deduced amino acid sequence of E2-C indicates that it is a new UBC family member. Bacterially expressed recombinant E2-C is active in in vitro cyclin ubiquitination assays, where it exhibits the same substrate specificities seen with native E2-C. These results demonstrate that E2-C is not a homolog of UBC4 or UBC9, proteins previously suggested to be involved in cyclin ubiquitination, but is a new UBC family member with unique properties.

Isolation of a chloroplast N,N'-dicyclohexylcarbodiimide-binding proteolipid, active in proton translocation.

The N,N'-dicyclohexylcarbodiimide-binding proteolipid from lettuce chloroplast membranes has been purified by a novel, rapid technique involving I-butanol extraction and ether precipitation. Reconstitution of this proteolipid into liposomes composed of chloroplast lipids and subsequent incorporation of bacteriorhodopsin resulted in the formation of liposomes exhibiting a light-dependent accumulation of protons. This accumulation was significantly enhanced upon addition of N,N'-dicyclohexylcarbodiimide at concentrations similar to those that inhibit chloroplast adenosinetriphosphatase activity. Radioactively labeled N,N'-dicyclohexylcarbodiimide was found to be incorporated essentially into the proteolipid of the reconstituted liposomes. These results suggest that the functional unit responsible for proton channeling in the chloroplast membrane has been isolated and reconstituted in the native state.

Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation.

The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin-proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin-proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK-)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor.

Components of a system that ligates cyclin to ubiquitin and their regulation by the protein kinase cdc2.

Cyclin B, a positive regulatory subunit of the cdc2 protein kinase complex, is synthesized across the cell cycle and then rapidly degraded at the end of mitosis. Degradation of cyclin B is triggered by increased levels of active cdc2 and is required for exit from mitosis. It was shown previously that cyclin degradation is carried out by the ubiquitin system, but the components responsible for the specificity and regulation of cyclin-ubiquitin ligation have not been identified. The formation of ubiquitin-protein conjugates usually requires the sequential action of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-carrier protein (E2), and a ubiquitin-protein ligase (E3). In this work we employed a fractionation approach to identify the components of a clam oocyte system responsible for specific ubiquitination of cyclin and to determine which components are regulated by cdc2. Experimental conditions were established under which a fusion protein containing an amino-terminal fragment of cyclin B is ligated to ubiquitin only in extracts from M-phase but not from interphase cells. Fractionation of M-phase extracts by DEAE-cellulose and high speed centrifugation yielded three fractions that were all required for cell cycle stage-specific cyclin-ubiquitin ligation. Only one of these fractions could be replaced by a previously known enzyme of the ubiquitin system, E1. A second fraction contained a novel species of E2, termed E2-C, which acts in the ligation of ubiquitin to cyclin but not to other endogenous proteins. A third component is associated with particulate material. Whereas E2-C from either M-phase or interphase extracts is active, the particulate component is active only in M-phase. Incubation of the particulate fraction from interphase cells with the protein kinase cdc2 activates it for cyclin-ubiquitin ligation, after a lag of about 30 min. These findings suggest that the particulate fraction may contain an E3 enzyme that acts on cyclin, as well as additional factors activated by cdc2.

SCF(beta)(-TrCP) ubiquitin ligase-mediated processing of NF-kappaB p105 requires phosphorylation of its C-terminus by IkappaB kinase.

Processing of the p105 precursor to form the active subunit p50 of the NF-kappaB transcription factor is a unique case in which the ubiquitin system is involved in limited processing rather than in complete destruction of the target substrate. A glycine-rich region along with a downstream acidic domain have been demonstrated to be essential for processing. Here we demonstrate that following IkappaB kinase (IkappaK)-mediated phosphorylation, the C-terminal domain of p105 (residues 918-934) serves as a recognition motif for the SCF(beta)(-TrCP) ubiquitin ligase. Expression of IkappaKbeta dramatically increases processing of wild-type p105, but not of p105-Delta918-934. Dominant-negative beta-TrCP inhibits IkappaK-dependent processing. Furthermore, the ligase and wild-type p105 but not p105-Delta918-934 associate physically following phosphorylation. In vitro, SCF(beta)(-TrCP) specifically conjugates and promotes processing of phosphorylated p105. Importantly, the TrCP recognition motif in p105 is different from that described for IkappaBs, beta-catenin and human immunodeficiency virus type 1 Vpu. Since p105-Delta918-934 is also conjugated and processed, it appears that p105 can be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs.