Frizzled7 as an emerging target for cancer therapy

Wnt proteins are secreted glycoproteins that bind to the N-terminal extra-cellular cysteine-rich domain of the Frizzled (Fzd) receptor family. The Fzd receptors can respond to Wnt proteins in the presence of Wnt co-receptors to activate the canonical and non-canonical Wnt pathways. Recent studies indicated that, among the Fzd family, Fzd7 is the Wnt receptor most commonly upregulated in a variety of cancers including colorectal cancer, hepatocellular carcinoma and triple negative breast cancer. Fzd7 plays an important role in stem cell biology and cancer development and progression. In addition, it has been demonstrated that siRNA knockdown of Fzd7, the anti-Fzd7 antibody or the extracellular peptide of Fzd7 (soluble Fzd7 peptide) displayed anti-cancer activity in vitro and in vivo mainly due to the inhibition of the canonical Wnt signaling pathway. Furthermore, pharmacological inhibition of Fzd7 by small interfering peptides or a small molecule inhibitor suppressed β-catenin-dependent tumor cell growth. Therefore, targeted inhibition of Fzd7 represents a rational and promising new approach for cancer therapy.     

Muscle RING-finger protein-1 (MuRF1) as a connector of muscle energy metabolism and protein synthesis

During pathophysiological muscle wasting, a family of ubiquitin ligases, including muscle RING-finger protein-1 (MuRF1), has been proposed to trigger muscle protein degradation via ubiquitination. Here, we characterized skeletal muscles from wild-type (WT) and MuRF1 knockout (KO) mice under amino acid (AA) deprivation as a model for physiological protein degradation, where skeletal muscles altruistically waste themselves to provide AAs to other organs. When WT and MuRF1 KO mice were fed a diet lacking AA, MuRF1 KO mice were less susceptible to muscle wasting, for both myocardium and skeletal muscles. Under AA depletion, WT mice had reduced muscle protein synthesis, while MuRF1 KO mice maintained nonphysiologically elevated levels of skeletal muscle protein de novo synthesis. Consistent with a role of MuRF1 for muscle protein turnover during starvation, the concentrations of essential AAs, especially branched-chain AAs, in the blood plasma significantly decreased in MuRF1 KO mice under AA deprivation. To clarify the molecular roles of MuRF1 for muscle metabolism during wasting, we searched for MuRF1-associated proteins using pull-down assays and mass spectrometry. Muscle-type creatine kinase (M-CK), an essential enzyme for energy metabolism, was identified among the interacting proteins. Coexpression studies revealed that M-CK interacts with the central regions of MuRF1 including its B-box domain and that MuRF1 ubiquitinates M-CK, which triggers the degradation of M-CK via proteasomes. Consistent with MuRF1's role of adjusting CK activities in skeletal muscles by regulating its turnover in vivo, we found that CK levels were significantly higher in the MuRF1 KO mice than in WT mice. Glucocorticoid modulatory element binding protein-1 and 3-hydroxyisobutyrate dehydrogenase, previously identified as potential MuRF1-interacting proteins, were also ubiquitinated MuRF1-dependently. Taken together, these data suggest that, in a multifaceted manner, MuRF1 participates in the regulation of AA metabolism, including the control of free AAs and their supply to other organs under catabolic conditions, and in the regulation of ATP synthesis under metabolic-stress conditions where MuRF1 expression is induced.     

Yeast surviving factor Svf1 as a new interacting partner, regulator and in vitro substrate of protein kinase CK2

Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant CK2α (K _m 0.35 μM) and CK2α′ (K _m 0.18 μM) as well as CK2 holoenzyme (K _m 1.1 μM). Different K _m values argue that CK2β(β′) subunit has an inhibitory effect on the activity of both CK2α and CK2α′ towards surviving factor Svf1. Reconstitution of α′₂ββ′ isoform of CK2 holoenzyme shows that β/β′ subunits have regulatory effect depending on the kind of CK2 catalytic subunit. This effect was not observed in the case of α₂ββ′ isoform, which may be due to interaction between Svf1 and regulatory CK2β subunit (shown by co-immunoprecipitation experiments). Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K¹⁹⁹EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED²⁴⁸ of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation of cell survival pathways.     

The kinesin I family member KIF5C is a novel substrate for protein kinase CK2

Protein kinase CK2 is ubiquitously expressed. The holoenzyme is composed of two catalytic α- or α′-subunits and two regulatory β-subunits but evidence is accumulating that the subunits can function independently. The composition of the holoenzyme as well as the expression of the individual subunits varies in different tissues, with high expression of CK2α′ in testis and brain. CK2 phosphorylates a number of different substrates which are implicated in basal cellular processes such as proliferation and survival of cells. Here, we report a new substrate, KIF5C, which is a member of the kinesin 1 family of motor neuron proteins. Phosphorylation of KIF5C was demonstrated in vitro and in vivo. Using deletion mutants, a peptide library, and mutation analysis a phosphorylation site for CK2 was mapped to amino acid 338 which is located in the non-motor domain of KIF5C. Interestingly, KIF5C is phosphorylated by holoenzymes composed of CK2α/CK2β and CK2α′/CK2β as well as by CK2α′ alone but not by CK2α alone.     

Inducible expression of the regulatory protein kinase CK2β subunit: Incorporation into complexes with catalytic CK2 subunits and re‐examination of the effects of CK2β on cell proliferation

The regulatory subunit of protein kinase CK2, designated CK2β, exists both free in cells and in complexes with the CK2 catalytic subunits. Growing evidence suggests that CK2β has functions dependent and independent of the CK2 catalytic subunits. There have been indications that CK2β has functions associated with DNA damage responses and in the control of cell proliferation. For example, transient and stable constitutive overexpression of CK2β in mammalian cells was previously shown to perturb cell cycle progression and to attenuate proliferation. To systematically investigate the molecular mechanisms responsible for these effects of CK2β on cell proliferation, we generated human osteosarcoma U2OS cell lines with tetracycline‐regulated expression of CK2β. Increased expression of CK2β results in increases in total cellular CK2 activity, but no changes in cell cycle profiles or proliferation. Furthermore, following exposure to ultraviolet radiation, p53 induction was identical regardless of the levels of CK2β in cells. Mouse 3T3‐L1 cells stably transfected with CK2β also showed no alterations in cell proliferation. The differences between these results and those previously reported emphasize the complex nature of CK2β and its cellular functions. Furthermore, these results indicate that increased expression of CK2β is not by itself sufficient to effect alterations in cell proliferation. J. Cell. Biochem. 84: 84–99, 2002. © 2001 Wiley‐Liss, Inc.     

Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells

The proapoptotic protein, prostate apoptosis response-4 (Par-4), acts as a tumor suppressor in prostate cancer cells. The serine/threonine kinase casein kinase 2 (CK2) has a well-reported role in prostate cancer resistance to apoptotic agents or anticancer drugs. However, the mechanistic understanding on how CK2 supports survival is far from complete. In this work, we demonstrate both in rat and humans that (i) Par-4 is a new substrate of the survival kinase CK2 and (ii) phosphorylation by CK2 impairs Par-4 proapoptotic functions. We also unravel different levels of CK2-dependent regulation of Par-4 between species. In rats, the phosphorylation by CK2 at the major site, S124, prevents caspase-mediated Par-4 cleavage (D123) and consequently impairs the proapoptotic function of Par-4. In humans, CK2 strongly impairs the apoptotic properties of Par-4, independently of the caspase-mediated cleavage of Par-4 (D131), by triggering the phosphorylation at residue S231. Furthermore, we show that human Par-4 residue S231 is highly phosphorylated in prostate cancer cells as compared with their normal counterparts. Finally, the sensitivity of prostate cancer cells to apoptosis by CK2 knockdown is significantly reversed by parallel knockdown of Par-4. Thus, Par-4 seems a critical target of CK2 that could be exploited for the development of new anticancer drugs.     

Evidence for allosteric effects on p53 oligomerization induced by phosphorylation

p53 is a tetrameric protein with a thermodynamically unstable deoxyribonucleic acid (DNA)‐binding domain flanked by intrinsically disordered regulatory domains that control its activity. The unstable and disordered segments of p53 allow high flexibility as it interacts with binding partners and permits a rapid on/off switch to control its function. The p53 tetramer can exist in multiple conformational states, any of which can be stabilized by a particular modification. Here, we apply the allostery model to p53 to ask whether evidence can be found that the “activating” C‐terminal phosphorylation of p53 stabilizes a specific conformation of the protein in the absence of DNA. We take advantage of monoclonal antibodies for p53 that measure indirectly the following conformations: unfolded, folded, and tetrameric. A double antibody capture enzyme linked‐immunosorbent assay was used to observe evidence of conformational changes of human p53 upon phosphorylation by casein kinase 2 in vitro. It was demonstrated that oligomerization and stabilization of p53 wild‐type conformation results in differential exposure of conformational epitopes PAb1620, PAb240, and DO12 that indicates a reduction in the “unfolded” conformation and increases in the folded conformation coincide with increases in its oligomerization state. These data highlight that the oligomeric conformation of p53 can be stabilized by an activating enzyme and further highlight the utility of the allostery model when applied to understanding the regulation of unstable and intrinsically disordered proteins.     

Phospho‐Proteomic Analysis of Cardiac Dyssynchrony and Resynchronization Therapy

Cardiac dyssynchrony arises from conduction abnormalities during heart failure and worsens morbidity and mortality. Cardiac resynchronization therapy (CRT) re‐coordinates contraction using bi‐ventricular pacing, but the cellular and molecular mechanisms involved remain largely unknown. The aim is to determine how dyssynchronous heart failure (HF_dys) alters the phospho‐proteome and how CRT interacts with this unique phospho‐proteome by analyzing Ser/Thr and Tyr phosphorylation. Phospho‐enriched myocardium from dog models of Control, HF_dys, and CRT is analyzed via MS. There were 209 regulated phospho‐sites among 1761 identified sites. Compared to Con and CRT, HF_dys is hyper‐phosphorylated and tyrosine phosphorylation is more likely to be involved in signaling that increased with HF_dys and was exacerbated by CRT. For each regulated site, the most‐likely targeting‐kinase is predicted, and CK2 is highly specific for sites that are “fixed” by CRT, suggesting activation of CK2 signaling occurs in HF_dys that is reversed by CRT, which is supported by western blot analysis. These data elucidate signaling networks and kinases that may be involved and deserve further study. Importantly, a possible role for CK2 modulation in CRT has been identified. This may be harnessed in the future therapeutically to compliment CRT, improving its clinical effects.     

Inactivation of glycogen synthase kinase 3β (GSK-3β) enhances mitochondrial biogenesis during myogenesis

Background

Mitochondrial biogenesis is crucial for myogenic differentiation and regeneration of skeletal muscle tissue and is tightly controlled by the peroxisome proliferator-activated receptor-γ co-activator 1 (PGC-1) signaling network. In the present study, we hypothesized that inactivation of glycogen synthase kinase (GSK)-3β, previously suggested to interfere with PGC-1 in non-muscle cells, potentiates PGC-1 signaling and the development of mitochondrial biogenesis during myogenesis, ultimately resulting in an enhanced myotube oxidative capacity.