E3 ligase EDD1/UBR5 is utilized by the HPV E6 oncogene to destabilize tumor suppressor TIP60.

Tat-interacting protein of 60 kDa (TIP60) is an essential lysine acetyltransferase implicated in transcription, DNA damage response and apoptosis. TIP60 protein expression is reduced in cancers. In cervical cancers, human papillomavirus (HPV) E6 oncogene targets cellular p53, Bak and some of the PDZ domain-containing proteins for proteasome-mediated degradation through E6AP ligase. Recently, E6 oncogene from high-risk and low-risk categories was also shown to target TIP60. However, the molecular mechanisms and whether destabilization of TIP60 contributes to HPV E6-mediated transformation remain unanswered. Our proteomic analyses revealed EDD1 (E3 identified by differential display), an E3 ligase generally overexpressed in cancers as a novel interacting partner of TIP60. By investigating protein turnover and ubiquitination assays, we show that EDD1 negatively regulates TIP60's stability through the proteasome pathway. Strikingly, HPV E6 uses this function of EDD1 to destabilize TIP60. Colony-formation assays and soft agar assays show that gain of function of TIP60 or depletion of EDD1 in HPV-positive cervical cancer cells significantly inhibits cell growth in vitro. This phenotype is strongly supported by the in-vivo studies where re-activation of TIP60 in cervical cancer cells dramatically reduces tumor formation. In summary, we have discovered a novel ligase through which E6 destabilizes TIP60. Currently, in the absence of an effective therapeutic vaccine for malignant cervical cancers, cervical cancer still remains to be a major disease burden. Hence, our studies implying a distinct tumor suppressor role for TIP60 in cervical cancers show that reactivation of TIP60 could be of therapeutic value.

TYK2 and JAK2 are substrates of protein-tyrosine phosphatase 1B.

The reversible tyrosine phosphorylation of proteins, modulated by the coordinated actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs), regulates the cellular response to a wide variety of stimuli. It is established that protein kinases possess discrete sets of substrates and that substrate recognition is often dictated by the presence of consensus phosphorylation sites. Here, we have extended this concept to the PTPs and demonstrated that (E/D)-pY-pY-(R/K) is a consensus substrate recognition motif for PTP1B. We have shown that JAK2 and TYK2 are substrates of PTP1B and that the substrate recognition site within theses kinases is similar to the site of dephosphorylation previously identified within the insulin receptor. A substrate-trapping mutant of PTP1B formed a stable interaction with JAK2 and TYK2 in response to interferon stimulation. Expression of wild type or substrate-trapping mutant PTP1B inhibited interferon-dependent transcriptional activation. Finally, mouse embryo fibroblasts deficient in PTP1B displayed subtle changes in tyrosine phosphorylation, including hyperphosphorylation of JAK2. The closely related JAK family member, JAK1, which does not match the consensus dephosphorylation site, was not recognized as a substrate. These data illustrate that PTP1B may be an important physiological regulator of cytokine signaling and that it may be possible to derive consensus substrate recognition motifs for other members of the PTP family, which may then be used to predict novel physiological substrates.

Glycosylation increases potassium channel stability and surface expression in mammalian cells.

N-linked glycosylation is not required for the cell surface expression of functional Shaker potassium channels in Xenopus oocytes (Santacruz-Toloza, L., Huang, Y., John, S. A., and Papazian, D. M. (1994) Biochemistry 33, 5607-5613). We have now investigated whether glycosylation increases the stability, cell surface expression, and proper folding of Shaker protein expressed in mammalian cells. The turnover rates of wild-type protein and an unglycosylated mutant (N259Q,N263Q) were compared in pulse-chase experiments. The wild-type protein was stable, showing little degradation after 48 h. In contrast, the unglycosylated mutant was rapidly degraded (t(1/2) = approximately 18 h). Lactacystin slowed the degradation of the mutant protein, implicating cytoplasmic proteasomes in its turnover. Rapid lactacystin-sensitive degradation could be conferred on wild-type Shaker by a glycosylation inhibitor. Expression of the unglycosylated mutant on the cell surface, assessed using immunofluorescence microscopy and biotinylation, was dramatically reduced compared with wild type. Folding and assembly were analyzed by oxidizing intersubunit disulfide bonds, which provides a fortuitous hallmark of the native structure. Surprisingly, formation of disulfide-bonded adducts was quantitatively similar in the wild-type and unglycosylated mutant proteins. Our results indicate that glycosylation increases the stability and cell surface expression of Shaker protein but has little effect on acquisition of the native structure.

PTEN controls tumor-induced angiogenesis.

Mutations of the tumor suppressor PTEN, a phosphatase with specificity for 3-phosphorylated inositol phospholipids, accompany progression of brain tumors from benign to the most malignant forms. Tumor progression, particularly in aggressive and malignant tumors, is associated with the induction of angiogenesis, a process termed the angiogenic switch. Therefore, we tested whether PTEN regulates tumor progression by modulating angiogenesis. U87MG glioma cells stably reconstituted with PTEN cDNA were tested for growth in a nude mouse orthotopic brain tumor model. We observed that the reconstitution of wild-type PTEN had no effect on in vitro proliferation but dramatically decreased tumor growth in vivo and prolonged survival in mice implanted intracranially with these tumor cells. PTEN reconstitution diminished phosphorylation of AKT within the PTEN-reconstituted tumor, induced thrombospondin 1 expression, and suppressed angiogenic activity. These effects were not observed in tumors reconstituted with a lipid phosphatase inactive G129E mutant of PTEN, a result that provides evidence that the lipid phosphatase activity of PTEN regulates the angiogenic response in vivo. These data provide evidence that PTEN regulates tumor-induced angiogenesis and the progression of gliomas to a malignant phenotype via the regulation of phosphoinositide-dependent signals.

Biochemical characterization and localization of the dual specificity kinase CLK1.

CLK1 was one of the first identified dual specificity kinases and is the founding member of the 'LAMMER' family of kinases. We have established the substrate site specificity of CLK1. We report here that truncation of the N terminus of CLK1 resulted in a dramatic increase in CLK1 enzymatic activity, indicating that the N terminus acts as a negative regulatory domain. The N-terminal truncation resulted in a 45-fold increase in V(max), suggesting that this domain does not contain a pseudo-substrate motif, but may act to conformationally constrain the catalytic activity of CLK1. Tyrosine phosphorylation has been proposed to be critical for CLK1 activity, however, CLK1 activity was unaffected by exposure to tyrosine phosphatases. Treatment of CLK1 with the serine/threonine specific phosphatase PP2A, resulted in a 2- to 6-fold increase in enzymatic activity. Incubation of CLK1 with tyrosine phosphatases in combination with PP2A abolished CLK1 activity. These data suggest that CLK1 is regulated by three distinct mechanisms that serve to both positively and negatively regulate CLK1 activity. CLK1 activity is positively regulated by phosphorylation on either tyrosine residues or serine/threonine residues, and is negatively regulated by steric constraints mediated by the N-terminal domain, as well as, by phosphorylation on a subset of serine/threonine residues within the catalytic domain. CLK1 mRNA is expressed at low levels in all tissues and cell lines examined. The full-length and truncated splice forms are expressed at roughly equivalent levels in most tissues. The ratio of the two splice variants of CLK1 can be altered by treatment with cycloheximide. CLK1 protein expression is limited to a small subset of highly localized neuronal populations in the rat brain. Contrary to previous studies using overexpression systems, we show that CLK1 protein is primarily found in the cytoplasm of these cells, with only a small fraction localized to the nucleus.

A point mutation in the maxi-K clone dSlo forms a high affinity site for charybdotoxin.

This work investigated the interaction of CTX with two cloned analogues of the maxi-K channel, dSlo and hSlo. dSlo has been reported to be CTX insensitive. Single channel analysis revealed that dSlo was weakly blocked by the toxin, with a very high K(D) of 5.8 microM. The hSlo channels bound wild-type, recombinant CTX with high affinity and in a bimolecular fashion, and displayed a half-blocking concentration (K(D)) of 36 nM. A glutamate residue was substituted for the wild-type threonine at position 290 in dSlo. The mutant channel was expressed in COS-7 cells and reconstituted into lipid bilayers for single channel analysis. The mutant channel bound wild-type, recombinant CTX with high affinity and in a bimolecular fashion, and displayed a half-blocking concentration (K(D)) of 23 nM. Changing just one residue from threonine to glutamate at position 290 in dSlo changed the affinity of the channel's CTX-receptor over 100-fold. Kinetic analysis revealed that this large increase in affinity was due to decreasing the dissociation rate of the toxin. These results suggest that a CTX receptor does exist in the dSlo channel mouth and that the threonine at position 290 destabilizes the toxin on the binding site.

Molecular basis for the dephosphorylation of the activation segment of the insulin receptor by protein tyrosine phosphatase 1B.

The protein tyrosine phosphatase PTP1B is responsible for negatively regulating insulin signaling by dephosphorylating the phosphotyrosine residues of the insulin receptor kinase (IRK) activation segment. Here, by integrating crystallographic, kinetic, and PTP1B peptide binding studies, we define the molecular specificity of this reaction. Extensive interactions are formed between PTP1B and the IRK sequence encompassing the tandem pTyr residues at 1162 and 1163 such that pTyr-1162 is selected at the catalytic site and pTyr-1163 is located within an adjacent pTyr recognition site. This selectivity is attributed to the 70-fold greater affinity for tandem pTyr-containing peptides relative to mono-pTyr peptides and predicts a hierarchical dephosphorylation process. Many elements of the PTP1B-IRK interaction are unique to PTP1B, indicating that it may be feasible to generate specific, small molecule inhibitors of this interaction to treat diabetes and obesity.

The CLK family kinases, CLK1 and CLK2, phosphorylate and activate the tyrosine phosphatase, PTP-1B.

The protein-tyrosine phosphatase PTP-1B is an important regulator of intracellular protein tyrosine phosphorylation, and is itself regulated by phosphorylation. We report that PTP-1B and its yeast analog, YPTP, are phosphorylated and activated by members of the CLK family of dual specificity kinases. CLK1 and CLK2 phosphorylation of PTP-1B in vitro activated the phosphatase activity approximately 3-5-fold using either p-nitrophenol phosphate, or tyrosine-phosphorylated myelin basic protein as substrates. Co-expression of CLK1 or CLK2 with PTP-1B in HEK 293 cells led to a 2-fold stimulation of phosphatase activity in vivo. Phosphorylation of PTP-1B at Ser(50) by CLK1 or CLK2 is responsible for its enzymatic activation. These findings suggest that phosphorylation at Ser(50) by serine threonine kinases may regulate the activation of PTP-1B in vivo. We also show that CLK1 and CLK2 phosphorylate and activate the S. cerevisiae PTP-1B family member, YPTP1. CLK1 phosphorylation of YPTP1 led to a 3-fold stimulation of phosphatase activity in vitro. We demonstrate that CLK phosphorylation of Ser(83) on YPTP1 is responsible for the activation of this enzyme. These findings demonstrate that the CLK kinases activate PTP-1B family members, and this phosphatase may be an important cellular target for CLK action.

The PTEN/MMAC1 tumor suppressor induces cell death that is rescued by the AKT/protein kinase B oncogene.

PTEN/MMAC1 is a tumor suppressor gene that is mutated in a variety of cancers. PTEN encodes a phosphatase that recognizes phosphoprotein substrates and the phospholipid, phosphatidylinositol-3,4,5-triphosphate. PTEN inhibited cell growth and/or colony formation in all of the epithelial lines tested with one exception. The decrease in cellular proliferation was associated with an induction of apoptosis and an inhibition of signaling through the phosphatidylinositol 3'-kinase pathway. Akt/protein kinase B, a gene whose antiapoptotic function is regulated by phosphatidylinositol-3,4,5-triphosphate, was able to rescue cells from PTEN-dependent death. PTEN, therefore, appears to suppress tumor growth by regulating phosphatidylinositol 3'-kinase signaling.

The lipid phosphatase activity of PTEN is critical for its tumor supressor function.

Since their discovery, protein tyrosine phosphatases have been speculated to play a role in tumor suppression because of their ability to antagonize the growth-promoting protein tyrosine kinases. Recently, a tumor suppressor from human chromosome 10q23, called PTEN or MMAC1, has been identified that shares homology with the protein tyrosine phosphatase family. Germ-line mutations in PTEN give rise to several related neoplastic disorders, including Cowden disease. A key step in understanding the function of PTEN as a tumor suppressor is to identify its physiological substrates. Here we report that a missense mutation in PTEN, PTEN-G129E, which is observed in two Cowden disease kindreds, specifically ablates the ability of PTEN to recognize inositol phospholipids as a substrate, suggesting that loss of the lipid phosphatase activity is responsible for the etiology of the disease. Furthermore, expression of wild-type or substrate-trapping forms of PTEN in HEK293 cells altered the levels of the phospholipid products of phosphatidylinositol 3-kinase and ectopic expression of the phosphatase in PTEN-deficient tumor cell lines resulted in the inhibition of protein kinase (PK) B/Akt and regulation of cell survival.

P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase.

Protein tyrosine phosphatases (PTPs) have long been thought to play a role in tumor suppression due to their ability to antagonize the growth promoting protein tyrosine kinases. Recently, a candidate tumor suppressor from 10q23, termed P-TEN, was isolated, and sequence homology was demonstrated with members of the PTP family, as well as the cytoskeletal protein tensin. Here we show that recombinant P-TEN dephosphorylated protein and peptide substrates phosphorylated on serine, threonine, and tyrosine residues, indicating that P-TEN is a dual-specificity phosphatase. In addition, P-TEN exhibited a high degree of substrate specificity, showing selectivity for extremely acidic substrates in vitro. Furthermore, we demonstrate that mutations in P-TEN, identified from primary tumors, tumor cells lines, and a patient with Bannayan-Zonana syndrome, resulted in the ablation of phosphatase activity, demonstrating that enzymatic activity of P-TEN is necessary for its ability to function as a tumor suppressor.

Comparison of chromosomal DNA composing timeless in Drosophila melanogaster and D. virilis suggests a new conserved structure for the TIMELESS protein.

Two proteins, TIM and PER, physically interact to control circadian cycles of tim and per transcription in Drosophila melanogaster. In the present study the structure of TIM protein expressed by D. virilis was determined by isolation and sequence analysis of genomic DNA (gDNA) corresponding to the D. virilis tim locus (v tim ). Comparison of v tim and m tim gDNA revealed high conservation of the TIM protein. This contrasts with poor sequence conservation previously observed for the TIM partner protein PER in these species. Inspection of the v tim sequence suggests an alternative structure for most TIM proteins. Sequences forming an intron in a previously characterized D. melanogaster tim cDNA appear to be most often translated to produce a longer TIM protein in both species. The N-terminal sequence of vTIM and sequence analysis of genomic DNA from several strains of D. melanogaster suggest that only one of two possible translation initiation sites found in tim mRNA is sufficient to generate circadian rhythms in D. melanogaster. TIM translation may be affected by multiple AUG codons that appear to have been conserved in sequences composing the 5'-untranslated tim mRNA leader.

Light-induced degradation of TIMELESS and entrainment of the Drosophila circadian clock.

Two genes, period (per) and timeless (tim), are required for production of circadian rhythms in Drosophila. The proteins encoded by these genes (PER and TIM) physically interact, and the timing of their association and nuclear localization is believed to promote cycles of per and tim transcription through an autoregulatory feedback loop. Here it is shown that TIM protein may also couple this molecular pacemaker to the environment, because TIM is rapidly degraded after exposure to light. TIM accumulated rhythmically in nuclei of eyes and in pacemaker cells of the brain. The phase of these rhythms was differentially advanced or delayed by light pulses delivered at different times of day, corresponding with phase shifts induced in the behavioral rhythms.

A light-entrainment mechanism for the Drosophila circadian clock.

Biochemical studies indicate that the Drosophila timeless protein (Tim) is a stoichiometric partner of the period protein (Per) in fly head extracts. A Per-Tim heterodimeric complex explains the reciprocal autoregulation of the proteins on transcription. The complex is under clock control, and many circadian features of the Tim cycle resemble those of the Per cycle. However, Tim is rapidly degraded in the early morning or in response to light, releasing Per from the complex. The Per-Tim complex is a functional unit of the Drosophila circadian clock, and Tim degradation may be the initial response of the clock to light.

Positional cloning and sequence analysis of the Drosophila clock gene, timeless.

The Drosophila genes timeless (tim) and period (per) interact, and both are required for production of circadian rhythms. Here the positional cloning and sequencing of tim are reported. The tim gene encodes a previously uncharacterized protein of 1389 amino acids, and possibly another protein of 1122 amino acids. The arrhythmic mutation tim01 is a 64-base pair deletion that truncates TIM to 749 amino acids. Absence of sequence similarity to the PER dimerization motif (PAS) indicates that direct interaction between PER and TIM would require a heterotypic protein association.

Isolation of timeless by PER protein interaction: defective interaction between timeless protein and long-period mutant PERL.

The period (per) gene likely encodes a component of the Drosophila circadian clock. Circadian oscillations in the abundance of per messenger RNA and per protein (PER) are thought to arise from negative feedback control of per gene transcription by PER. A recently identified second clock locus, timeless (tim), apparently regulates entry of PER into the nucleus. Reported here are the cloning of complementary DNAs derived from the tim gene in a two-hybrid screen for PER-interacting proteins and the demonstration of a physical interaction between the tim protein (TIM) and PER in vitro. A restricted segment of TIM binds directly to a part of the PER dimerization domain PAS. PERL, a mutation that causes a temperature-sensitive lengthening of circadian period and a temperature-sensitive delay in PER nuclear entry, exhibits a temperature-sensitive defect in binding to TIM. These results suggest that the interaction between TIM and PER determines the timing of PER nuclear entry and therefore the duration of part of the circadian cycle.

Rhythmic expression of timeless: a basis for promoting circadian cycles in period gene autoregulation.

The clock gene timeless (tim) is required for circadian rhythmicity in Drosophila. The accumulation of tim RNA followed a circadian rhythm, and the phase and period of the tim RNA rhythm were indistinguishable from those that have been reported for per. The tim RNA oscillations were found to be dependent on the presence of PER and TIM proteins, which demonstrates feedback control of tim by a mechanism previously shown to regulate per expression. The cyclic expression of tim appears to dictate the timing of PER protein accumulation and nuclear localization, suggesting that tim promotes circadian rhythms of per and tim transcription by restricting per RNA and PER protein accumulation to separate times of day.