Protein phosphatase 2A: a panoply of enzymes.

Protein phosphatase 2A describes an extended family of intracellular protein serine/threonine phosphatases sharing a common catalytic subunit that regulates a variety of processes by means of diverse regulatory subunits. During the past year, studies have shown that protein phosphatase 2A influences events ranging from the initiation of DNA replication to vertebrate axis formation to apoptosis.

Clathrin-coated vesicle assembly polypeptides: physical properties and reconstitution studies with brain membranes.

The assembly polypeptides are an integral component of coated vesicles and may mediate the linkage of clathrin to the vesicle membrane. We have purified assembly polypeptides in milligram quantities from bovine brain by an improved procedure. Hydrodynamic and chemical crosslinking studies indicate that the protein is an asymmetric heterotetramer with a molecular weight of 252,000, containing two subunits of Mr 98,000-115,000, one subunit of 52,000, and one subunit of 16,000. Two-dimensional peptide maps of the subunits show that the 16- and 52-kD polypeptides are not derived from the higher molecular weight species, and that the group of bands at 98-115 kD are related. Electron microscopic visualization shows an essentially globular protein with one or two knob-like tails. We demonstrate a specific membrane protein binding site for 125I-labeled assembly polypeptides in 0.1 N sodium hydroxide-extracted bovine brain membranes based on the following criteria: (a) binding is displaceable by unlabeled ligand, (b) the binding site is destroyed by protease treatment of the membranes, and (c) the distribution of binding between vesicle-depleted membranes and coated vesicle membranes parallels the in vivo localization of assembly polypeptides and clathrin. This binding site is likely to be an integral membrane protein because (a) it is enriched in the sodium hydroxide-extracted membranes stripped of most of their peripheral membrane proteins, and (b) the binding site is partially extracted by 0.5% Triton X-100. A similar binding site appears to be present in coated vesicles. Clathrin binds to the hydroxide-stripped membranes in an assembly polypeptides dependent manner, and this binding is diminished by Triton extraction of the membranes. This assay may aid in identification of the membrane receptor for the assembly polypeptides.

Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A.

Dysregulation of Wnt-beta-catenin signaling disrupts axis formation in vertebrate embryos and underlies multiple human malignancies. The adenomatous polyposis coli (APC) protein, axin, and glycogen synthase kinase 3beta form a Wnt-regulated signaling complex that mediates the phosphorylation-dependent degradation of beta-catenin. A protein phosphatase 2A (PP2A) regulatory subunit, B56, interacted with APC in the yeast two-hybrid system. Expression of B56 reduced the abundance of beta-catenin and inhibited transcription of beta-catenin target genes in mammalian cells and Xenopus embryo explants. The B56-dependent decrease in beta-catenin was blocked by oncogenic mutations in beta-catenin or APC, and by proteasome inhibitors. B56 may direct PP2A to dephosphorylate specific components of the APC-dependent signaling complex and thereby inhibit Wnt signaling.

An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome.

Familial advanced sleep phase syndrome (FASPS) is an autosomal dominant circadian rhythm variant; affected individuals are "morning larks" with a 4-hour advance of the sleep, temperature, and melatonin rhythms. Here we report localization of the FASPS gene near the telomere of chromosome 2q. A strong candidate gene (hPer2), a human homolog of the period gene in Drosophila, maps to the same locus. Affected individuals have a serine to glycine mutation within the casein kinase Iepsilon (CKIepsilon) binding region of hPER2, which causes hypophosphorylation by CKIepsilon in vitro. Thus, a variant in human sleep behavior can be attributed to a missense mutation in a clock component, hPER2, which alters the circadian period.

Oncogenic RAS-induced CK1α drives nuclear FOXO proteolysis.

Evasion of forkhead box O (FOXO) family of longevity-related transcription factors-mediated growth suppression is necessary to promote cancer development. Since somatic alterations or mutations and transcriptional dysregulation of the FOXO genes are infrequent in human cancers, it remains unclear how these tumour suppressors are eliminated from cancer cells. The protein stability of FOXO3A is regulated by Casein Kinase 1 alpha (CK1α) in an oncogenic RAS-specific manner, but whether this mode of regulation extends to related FOXO family members is unknown. Here we report that CK1α similarly destabilizes FOXO4 in RAS-mutant cells by phosphorylation at serines 265/268. The CK1α-dependent phosphoregulation of FOXO4 is primed, in part, by the PI3K/AKT effector axis of oncogenic RAS signalling. In addition, mutant RAS coordinately elevates proteasome subunit expression and proteolytic activity to eradicate nuclear FOXO4 proteins from RAS-mutant cancer cells. Importantly, dual inhibition of CK1α and the proteasome synergistically inhibited the growth of multiple RAS-mutant human cancer cell lines of diverse tissue origin by blockade of nuclear FOXO4 degradation and induction of caspase-dependent apoptosis. Our findings challenge the current paradigm that nuclear export regulates the proteolysis of FOXO3A/4 tumour suppressors in the context of cancer and illustrates how oncogenic RAS-mediated degradation of FOXOs, via post-translational mechanisms, blocks these important tumour suppressors.

Control of simian virus 40 DNA replication by the HeLa cell nuclear kinase casein kinase I.

The initiation of simian virus 40 (SV40) DNA replication is regulated by the phosphorylation state of the viral initiator protein, large T antigen. We describe the purification from HeLa cell nuclei of a 35-kDa serine/threonine protein kinase that phosphorylates T antigen at sites that are phosphorylated in vivo and thereby inhibits its ability to initiate SV40 DNA replication. The inhibition of both origin unwinding and DNA replication by the kinase is reversed by protein phosphatase 2A. As determined by molecular weight, substrate specificity, autophosphorylation, immunoreactivity, and limited sequence analysis, this kinase appears to be identical to casein kinase I, a ubiquitous serine/threonine protein kinase that is closely related to a yeast kinase involved in DNA metabolism. The HeLa cell phosphorylation cycle that controls the initiation of SV40 DNA replication may also play a role in cellular DNA metabolism.

Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A.

The catalytic subunit of protein phosphatase 2A (PP2Ac) stimulates the initiation of replication of simian virus 40 DNA in vitro by dephosphorylating T antigen at specific phosphoserine residues (K. H. Scheidtmann, D. M. Virshup, and T. J. Kelly, J. Virol. 65:2098-2101, 1991). To better define the biochemical mechanism responsible for this stimulation, we investigated the effect of PP2Ac on the interaction of T antigen with wild-type and mutant origins of replication. Analysis of the binding of T antigen to the wild-type origin as a function of protein concentration revealed that binding occurs in two relatively discrete steps: the assembly of a T-antigen hexamer on one half-site of the origin, followed by the assembly of the second hexamer on the other half-site. The major effect of PP2Ac was to stimulate binding of the second hexamer, so that the binding reaction became much more cooperative. This observation suggests that dephosphorylation of T antigen by PP2Ac primarily affects interactions between the two hexamers bound to the origin. Pretreatment with PP2Ac increased the ability of the bound T antigen to unwind the origin of replication but had no effect on the intrinsic helicase activity of the protein. Thus, dephosphorylation of PP2Ac appears to increase the efficiency of the initial opening of the origin by T antigen. An insertion mutation at the dyad axis in the simian virus 40 origin, which altered the structural relationship of the two halves of the origin, abolished the effect of the phosphatase on the cooperativity of binding and completely prevented origin unwinding. These findings suggest that the ability of T antigen to open the viral origin of DNA replication is critically dependent on the appropriate functional interactions between T-antigen hexamers and that these interactions are regulated by the phosphorylation state of the viral initiator protein.

Nuclear entry of the circadian regulator mPER1 is controlled by mammalian casein kinase I epsilon.

The molecular oscillator that keeps circadian time is generated by a negative feedback loop. Nuclear entry of circadian regulatory proteins that inhibit transcription from E-box-containing promoters appears to be a critical component of this loop in both Drosophila and mammals. The Drosophila double-time gene product, a casein kinase I epsilon (CKIepsilon) homolog, has been reported to interact with dPER and regulate circadian cycle length. We find that mammalian CKIepsilon binds to and phosphorylates the murine circadian regulator mPER1. Unlike both dPER and mPER2, mPER1 expressed alone in HEK 293 cells is predominantly a nuclear protein. Two distinct mechanisms appear to retard mPER1 nuclear entry. First, coexpression of mPER2 leads to mPER1-mPER2 heterodimer formation and cytoplasmic colocalization. Second, coexpression of CKIepsilon leads to masking of the mPER1 nuclear localization signal and phosphorylation-dependent cytoplasmic retention of both proteins. CKIepsilon may regulate mammalian circadian rhythm by controlling the rate at which mPER1 enters the nucleus.

Different oligomeric forms of protein phosphatase 2A activate and inhibit simian virus 40 DNA replication.

The ability of simian virus 40 (SV40) large T antigen to catalyze the initiation of viral DNA replication is regulated by its phosphorylation state. Previous studies have identified the free catalytic subunit of protein phosphatase 2A (PP2Ac) as the cellular phosphatase which can remove inhibitory phosphoryl groups from serines 120 and 123. The catalytic C subunit exists in the cell complexed with a 65-kDa A subunit and one of several B subunits. To determine if any of the holoenzymes could activate T antigen, we tested the ability of the heterodimeric AC and two heterotrimeric ABC forms to stimulate T-antigen function in unwinding the origin of SV40 DNA replication. Only free catalytic subunit C and the heterotrimeric form with a 72-kDa B subunit (PP2A-T72) could stimulate T-antigen-dependent origin unwinding. Both the dimeric form (PP2A-D) and the heterotrimer with a 55-kDa B subunit (PP2A-T55) actively inhibited T-antigen function. We found that PP2A-T72 activated T antigen by dephosphorylating serines 120 and 123, while PP2A-D and PP2A-T55 inactivated T antigen by dephosphorylating the p34cdc2 target site, threonine 124. Thus, alterations in the subunit composition of PP2A holoenzymes have significant functional consequences for the initiation of in vitro SV40 DNA replication. The regulatory B subunits of PP2A may play a role in regulating SV40 DNA replication in infected cells as well.

Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways.

The cascade that culminates in macrometastases is thought to be mediated by phenotypic plasticity, including epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT and MET). Although there is substantial support for the role of EMT in driving cancer cell invasion and dissemination, much less is known about the importance of MET in the later steps of metastatic colonization. We created novel reporters, which integrate transcriptional and post-transcriptional regulation, to test whether MET is required for metastasis in multiple in vivo cancer models. In a model of carcinosarcoma, metastasis occurred via an MET-dependent pathway; however, in two prostate carcinoma models, metastatic colonization was MET independent. Our results provide evidence for both MET-dependent and MET-independent metastatic pathways.

Wnt addiction of genetically defined cancers reversed by PORCN inhibition.

Enhanced sensitivity to Wnts is an emerging hallmark of a subset of cancers, defined in part by mutations regulating the abundance of their receptors. Whether these mutations identify a clinical opportunity is an important question. Inhibition of Wnt secretion by blocking an essential post-translational modification, palmitoleation, provides a useful therapeutic intervention. We developed a novel potent, orally available PORCN inhibitor, ETC-1922159 (henceforth called ETC-159) that blocks the secretion and activity of all Wnts. ETC-159 is remarkably effective in treating RSPO-translocation bearing colorectal cancer (CRC) patient-derived xenografts. This is the first example of effective targeted therapy for this subset of CRC. Consistent with a central role of Wnt signaling in regulation of gene expression, inhibition of PORCN in RSPO3-translocated cancers causes a marked remodeling of the transcriptome, with loss of cell cycle, stem cell and proliferation genes, and an increase in differentiation markers. Inhibition of Wnt signaling by PORCN inhibition holds promise as differentiation therapy in genetically defined human cancers.

Casein kinase 1 regulates Sprouty2 in FGF-ERK signaling.

Sprouty2 (SPRY2) is a potent negative regulator of receptor tyrosine kinase signaling, and is implicated as a tumor suppressor. SPRY2 inhibits FGF-RAS-ERK signaling by binding to growth factor receptor bound protein 2 (GRB2) during fibroblast growth factor receptor (FGFR) activation, disrupting the GRB2-SOS (son of sevenless) complex that transduces signals from FGFR to RAS. SPRY2 binding to GRB2 is modulated by phosphorylation but the key regulatory kinase(s) are not known. Prior studies identified the frequent presence of CK1 phosphorylation motifs on SPRY2. We therefore tested if CK1 has a role in SPRY2 phosphorylation and function. Loss of CK1 binding and inhibition of CK1 activity by two structurally distinct small molecules abrogated SPRY2 inhibition of FGF-ERK signaling, leading to decreased SPRY2 interaction with GRB2. Moreover, CK1 activity and binding are necessary for SPRY2 inhibition of FGF-stimulated neurite outgrowth in PC12 cells. Consistent with its proposed role as an inhibitor of FGF signaling, we find that CSNK1E transcript abundance negatively correlates with FGF1/FGF7 message in human gastric cancer samples. Modulation of CK1 activity may be therapeutically useful in the treatment of FGF/SPRY2-related diseases.

Human casein kinase Idelta phosphorylation of human circadian clock proteins period 1 and 2.

Casein kinase Iepsilon (CKIepsilon), a central component of the circadian clock, interacts with and phosphorylates human period protein 1 (hPER1) [Keesler, G.A. et al. (2000) NeuroReport 5, 951-955]. A mutation in CKIepsilon causes a shortened circadian period in Syrian Golden hamster. We have now extended our previous studies to show that human casein kinase Idelta (hCKIdelta), the closest homologue to hCKIepsilon, associates with and phosphorylates hPER1 and causes protein instability. Furthermore, we observed that both hCKIdelta and hCKIepsilon phosphorylated and caused protein instability of human period 2 protein (hPER2). Immunohistochemical staining of rat brains demonstrates that CKIdelta protein is localized in the suprachiasmatic nuclei, the central location of the master clock. These results indicate that CKIdelta may play a role similar to CKIepsilon, suggesting that it may also be involved in regulating circadian rhythmicity by post-translation modification of mammalian clock proteins hPER1 and 2.

Focal encephalitis with enterovirus infections.

We report on four pediatric patients with Enterovirus infections who were admitted to the hospital with signs or symptoms of acute, focal encephalitis. All four experienced focal seizures. Each had a cerebrospinal fluid pleocytosis at the initial lumbar puncture. In all four patients the diagnosis of herpes simplex encephalitis was entertained. Each child improved spontaneously within a few days of admission to the hospital, and only one had residual neurologic abnormalities at the time of discharge. A brief review of these cases, and three additional cases from the literature, indicate that the enteroviruses, particularly the group A Coxsackieviruses, are rare causes of acute focal encephalitis in children and adolescents.

Casein kinase I: another cog in the circadian clockworks.

Multiple components of the circadian central clock are phosphoproteins, and it has become increasingly clear that posttranslational modification is an important regulator of circadian rhythm in diverse organisms, from dinoflagellates to humans. Genetic studies in Drosophila have identified double-time (dbt), a serine/threonine protein kinase that is highly homologous to human casein kinase I epsilon (CKIepsilon), as the first kinase linked to behavioral rhythms. Identification of a missense mutation in CKIepsilon as the tau mutation in the Syrian hamster places CKIepsilon within the core clock machinery in mammals. Most recently, identification of a phosphorylation site mutant of hPER2 in a family with an inherited circadian rhythm abnormality strongly suggests that PER2 is a physiologically relevant substrate of CKI. Phosphorylation may regulate multiple properties of clock proteins, including stability and intracellular localization.

Modulation of Wnt/ß-catenin signaling and proliferation by a ferrous iron chelator with therapeutic efficacy in genetically engineered mouse models of cancer.

Using a screen for Wnt/ß-catenin inhibitors, a family of 8-hydroxyquinolone derivatives with in vivo anti-cancer properties was identified. Analysis of microarray data for the lead compound N-((8-hydroxy-7-quinolinyl) (4-methylphenyl)methyl)benzamide (HQBA) using the Connectivity Map database suggested that it is an iron chelator that mimics the hypoxic response. HQBA chelates Fe(2+) with a dissociation constant of ∼10(-19) M, with much weaker binding to Fe(3+) and other transition metals. HQBA inhibited proliferation of multiple cell lines in culture, and blocked the progression of established spontaneous cancers in two distinct genetically engineered mouse models of mammary cancer, MMTV-Wnt1 and MMTV-PyMT mice, without overt toxicity. HQBA may inhibit an iron-dependent factor that regulates cell-type-specific ß-catenin-driven transcription. It inhibits cancer cell proliferation independently of its effect on ß-catenin signaling, as it works equally well in MMTV-PyMT tumors and diverse ß-catenin-independent cell lines. HQBA is a promising specific intracellular Fe(2+) chelator with activity against spontaneous mouse mammary cancers.

IC261 induces cell cycle arrest and apoptosis of human cancer cells via CK1δ/ɛ and Wnt/ß-catenin independent inhibition of mitotic spindle formation.

Casein kinase 1 delta and epsilon (CK1δ/ɛ) are key regulators of diverse cellular growth and survival processes including Wnt signaling, DNA repair and circadian rhythms. Recent studies suggest that they have an important role in oncogenesis. RNA interference screens identified CK1ɛ as a pro-survival factor in cancer cells in vitro and the CK1δ/ɛ-specific inhibitor IC261 is remarkably effective at selective, synthetic lethal killing of cancer cells. The recent development of the nanomolar CK1δ/ɛ-selective inhibitor, PF670462 (PF670) and the CK1ɛ-selective inhibitor PF4800567 (PF480) offers an opportunity to further test the role of CK1δ/ɛ in cancer. Unexpectedly, and unlike IC261, PF670 and PF480 were unable to induce cancer cell death. PF670 is a potent inhibitor of CK1δ/ɛ in cells; nanomolar concentrations are sufficient to inhibit CK1δ/ɛ activity as measured by repression of intramolecular autophosphorylation, phosphorylation of disheveled2 proteins and Wnt/ß-catenin signaling. Likewise, small interfering RNA knockdown of CK1δ and CK1ɛ reduced Wnt/ß-catenin signaling without affecting cell viability, further suggesting that CK1δ/ɛ inhibition may not be relevant to the IC261-induced cell death. Thus, while PF670 is a potent inhibitor of Wnt signaling, it only modestly inhibits cell proliferation. In contrast, while sub-micromolar concentrations of IC261 neither inhibited CK1δ/ɛ kinase activity nor blocked Wnt/ß-catenin signaling in cancer cells, it caused a rapid induction of prometaphase arrest and subsequent apoptosis in multiple cancer cell lines. In a stepwise transformation model, IC261-induced killing required both overactive Ras and inactive p53. IC261 binds to tubulin with an affinity similar to colchicine and is a potent inhibitor of microtubule polymerization. This activity accounts for many of the diverse biological effects of IC261 and, most importantly, for its selective cancer cell killing.

Reversible protein phosphorylation regulates circadian rhythms.

Protein phosphorylation regulates the period of the circadian clock within mammalian cells. Circadian rhythms are an approximately 24-hour cycle that regulates key biological processes. Daily fluctuations of wakefulness, stress hormones, lipid metabolism, immune function, and the cell division cycle are controlled by the molecular clocks that function throughout our bodies. Mutations in regulatory components of the clock can shorten or lengthen the timing of the rhythms and have significant physiological consequences. The clock is formed by a negative feedback loop of transcription, translation, and inhibition of transcription. The precision of clock timing is controlled by protein kinases and phosphatases. Casein kinase Iepsilon is a protein kinase that regulates the circadian clock by periodic phosphorylation of the proteins PER1 and PER2, controlling their stability and localization. The role of phosphorylation in regulating PER function in the clock has been explored in detail. Quantitative modeling has proven to be very useful in making important predictions about how changes in phosphorylation alter the clock's behavior. Quantitative data from biological studies can be used to refine the quantitative model and make additional testable predictions. A detailed understanding of how reversible protein phosphorylation regulates circadian rhythms and a detailed quantitative model that makes clear, testable, and accurate predictions about the clock and how we may manipulate it can have important benefits for human health. Pharmacological manipulation of rhythms could mitigate stress from jet lag, shift work, and perhaps even seasonal affective disorder.