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International Journal of Molecular... Jun 2022Casein kinase 2 (CK2) is a ubiquitously expressed serine/threonine kinase and is upregulated in human obesity. CX-4945 (Silmitasertib) is a CK2 inhibitor with...
Casein kinase 2 (CK2) is a ubiquitously expressed serine/threonine kinase and is upregulated in human obesity. CX-4945 (Silmitasertib) is a CK2 inhibitor with anti-cancerous and anti-adipogenic activities. However, the anti-adipogenic and pro-lipolytic effects and the mode of action of CX-4945 in (pre)adipocytes remain elusive. Here, we explored the effects of CX-4945 on adipogenesis and lipolysis in differentiating and differentiated 3T3-L1 cells, a murine preadipocyte cell line. CX-4945 at 15 μM strongly reduced lipid droplet (LD) accumulation and triglyceride (TG) content in differentiating 3T3-L1 cells, indicating the drug's anti-adipogenic effect. Mechanistically, CX-4945 reduced the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and perilipin A in differentiating 3T3-L1 cells. Strikingly, CX-4945 further increased the phosphorylation levels of cAMP-activated protein kinase (AMPK) and liver kinase B-1 (LKB-1) while decreasing the intracellular ATP content in differentiating 3T3-L1 cells. In differentiated 3T3-L1 cells, CX-4945 had abilities to stimulate glycerol release and elevate the phosphorylation levels of hormone-sensitive lipase (HSL), pointing to the drug's pro-lipolytic effect. In addition, CX-4945 induced the activation of extracellular signal-regulated kinase-1/2 (ERK-1/2), and PD98059, an inhibitor of ERK-1/2, attenuated the CX4945-induced glycerol release and HSL phosphorylation in differentiated 3T3-L1 cells, indicating the drug's ERK-1/2-dependent lipolysis. In summary, this investigation shows that CX-4945 has strong anti-adipogenic and pro-lipolytic effects on differentiating and differentiated 3T3-L1 cells, mediated by control of the expression and phosphorylation levels of CK2, C/EBP-α, PPAR-γ, FAS, ACC, perilipin A, AMPK, LKB-1, ERK-1/2, and HSL.
Topics: 3T3-L1 Cells; AMP-Activated Protein Kinases; Adipogenesis; Animals; CCAAT-Enhancer-Binding Protein-alpha; Casein Kinase II; Cell Differentiation; Cyclic AMP-Dependent Protein Kinases; Glycerol; Humans; Lipolysis; Mice; Naphthyridines; PPAR gamma; Perilipin-1; Phenazines; Sterol Esterase
PubMed: 35806278
DOI: 10.3390/ijms23137274 -
International Journal of Molecular... Nov 2020The casein kinase 1 enzymes (CK1) form a family of serine/threonine kinases with seven CK1 isoforms identified in humans. The most important substrates of CK1 kinases... (Review)
Review
The casein kinase 1 enzymes (CK1) form a family of serine/threonine kinases with seven CK1 isoforms identified in humans. The most important substrates of CK1 kinases are proteins that act in the regulatory nodes essential for tumorigenesis of hematological malignancies. Among those, the most important are the functions of CK1s in the regulation of Wnt pathways, cell proliferation, apoptosis and autophagy. In this review we summarize the recent developments in the understanding of biology and therapeutic potential of the inhibition of CK1 isoforms in the pathogenesis of chronic lymphocytic leukemia (CLL), other non-Hodgkin lymphomas (NHL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and multiple myeloma (MM). CK1δ/ε inhibitors block CLL development in preclinical models via inhibition of WNT-5A/ROR1-driven non-canonical Wnt pathway. While no selective CK1 inhibitors have reached clinical stage to date, one dual PI3Kδ and CK1ε inhibitor, umbralisib, is currently in clinical trials for CLL and NHL patients. In MDS, AML and MM, inhibition of CK1α, acting via activation of p53 pathway, showed promising preclinical activities and the first CK1α inhibitor has now entered the clinical trials.
Topics: Animals; Antineoplastic Agents; Casein Kinase I; Hematologic Neoplasms; Humans; Molecular Targeted Therapy; Neoplastic Stem Cells; Wnt Signaling Pathway
PubMed: 33261128
DOI: 10.3390/ijms21239026 -
The Journal of Biological Chemistry 2021The study of extracellular phosphorylation was initiated in late 19th century when the secreted milk protein, casein, and egg-yolk protein, phosvitin, were shown to be... (Review)
Review
The study of extracellular phosphorylation was initiated in late 19th century when the secreted milk protein, casein, and egg-yolk protein, phosvitin, were shown to be phosphorylated. However, it took more than a century to identify Fam20C, which phosphorylates both casein and phosvitin under physiological conditions. This kinase, along with its family members Fam20A and Fam20B, defined a new family with altered amino acid sequences highly atypical from the canonical 540 kinases comprising the kinome. Fam20B is a glycan kinase that phosphorylates xylose residues and triggers peptidoglycan biosynthesis, a role conserved from sponges to human. The protein kinase, Fam20C, conserved from nematodes to humans, phosphorylates well over 100 substrates in the secretory pathway with overall functions postulated to encompass endoplasmic reticulum homeostasis, nutrition, cardiac function, coagulation, and biomineralization. The preferred phosphorylation motif of Fam20C is SxE/pS, and structural studies revealed that related member Fam20A allosterically activates Fam20C by forming a heterodimeric/tetrameric complex. Fam20A, a pseudokinase, is observed only in vertebrates. Loss-of-function genetic alterations in the Fam20 family lead to human diseases such as amelogenesis imperfecta, nephrocalcinosis, lethal and nonlethal forms of Raine syndrome with major skeletal defects, and altered phosphate homeostasis. Together, these three members of the Fam20 family modulate a diverse network of secretory pathway components playing crucial roles in health and disease. The overarching theme of this review is to highlight the progress that has been made in the emerging field of extracellular phosphorylation and the key roles secretory pathway kinases play in an ever-expanding number of cellular processes.
Topics: Casein Kinase I; Endoplasmic Reticulum; Extracellular Matrix Proteins; Homeostasis; Humans; Myocardium; Phosphorylation; Secretory Pathway; Signal Transduction; Structure-Activity Relationship; Substrate Specificity
PubMed: 33759783
DOI: 10.1016/j.jbc.2021.100267 -
ACS Chemical Biology May 2022O-GlcNAc is an essential carbohydrate modification that intersects with phosphorylation signaling pathways via crosstalk on protein substrates or by direct modification...
O-GlcNAc is an essential carbohydrate modification that intersects with phosphorylation signaling pathways via crosstalk on protein substrates or by direct modification of the kinases that write the phosphate modification. Casein kinase 2 alpha (CK2α), the catalytic subunit of the ubiquitously expressed and constitutively active kinase CK2, is modified by O-GlcNAc, but the effect of this modification on the phosphoproteome in cells is unknown. Here, we apply complementary targeted O-GlcNAc editors, nanobody-OGT and -splitOGA, to selectively write and erase O-GlcNAc from a tagged CK2α to measure the effects on the phosphoproteome in cells. These tools effectively and selectively edit the Ser347 glycosite on CK2α. Using quantitative phosphoproteomics, we report 51 phosphoproteins whose enrichment changes as a function of editing O-GlcNAc on CK2α, including HDAC1, HDAC2, ENSA, SMARCAD1, and PABPN1. Specific phosphosites on HDAC1 Ser393 and HDAC2 Ser394, both reported CK2 substrates, are significantly enhanced by O-GlcNAcylation of CK2α. These data will propel future studies on the crosstalk between O-GlcNAc and phosphorylation.
Topics: Acetylglucosamine; Casein Kinase II; N-Acetylglucosaminyltransferases; Phosphorylation; Proteome; Writing
PubMed: 35467332
DOI: 10.1021/acschembio.1c00987 -
Virulence Dec 2019Casein kinases are serine/threonine protein kinases that are evolutionarily conserved in yeast and humans and are involved in a range of important cellular processes....
Casein kinases are serine/threonine protein kinases that are evolutionarily conserved in yeast and humans and are involved in a range of important cellular processes. However, the biological functions of casein kinases in the fungus , the causal agent of destructive rice blast disease, are not characterized. Here, two casein kinases, and , were identified and targeted for replacement, but only was further characterized due to the possible nonviability of the deletion mutant. Disruption of caused pleiotropic defects in growth, conidiation, conidial germination, and appressorium formation and penetration, therefore resulting in reduced virulence in rice seedlings and barley leaves. Notably, the deletion triggered quick lipidation of MoAtg8 and degradation of the autophagic marker protein GFP-MoAtg8 under nitrogen starvation conditions, in contrast to the wild type, indicating that autophagy activity was negatively regulated by MoYck1. Furthermore, we found that HOPS (homotypic fusion and vacuolar protein sorting) subunit MoVps41, a putative substrate of MoYck1, was co-located with MoAtg8 and positively required for the degradation of MoAtg8-PE and GFP-MoAtg8. In addition, is also involved in the response to ionic hyperosmotic and heavy metal cation stresses. Taken together, our results revealed crucial roles of the casein kinase MoYck1 in regulating development, autophagy and virulence in .
Topics: Autophagy; Casein Kinases; Fungal Proteins; Gene Expression Regulation, Fungal; Gene Knockout Techniques; Hordeum; Magnaporthe; Mutation; Oryza; Plant Diseases; Spores, Fungal; Virulence; Virulence Factors
PubMed: 31392921
DOI: 10.1080/21505594.2019.1649588 -
The Journal of Biological Chemistry Jun 2022Aberrant activation or suppression of WNT/β-catenin signaling contributes to cancer initiation and progression, neurodegeneration, and bone disease. However, despite...
Aberrant activation or suppression of WNT/β-catenin signaling contributes to cancer initiation and progression, neurodegeneration, and bone disease. However, despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are considered exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and "dark." Here, we studied the function of the casein kinase 1γ (CSNK1γ) subfamily of human kinases and their roles in WNT signaling. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1γ1, CSNK1γ2, and CSNK1γ3 revealed numerous components of the β-catenin-dependent and β-catenin-independent WNT pathways. In gain-of-function experiments, we found that CSNK1γ3 but not CSNK1γ1 or CSNK1γ2 activated β-catenin-dependent WNT signaling, with minimal effect on other signaling pathways. We also show that within the family, CSNK1γ3 expression uniquely induced low-density lipoprotein receptor-related protein 6 phosphorylation, which mediates downstream WNT signaling transduction. Conversely, siRNA-mediated silencing of CSNK1γ3 alone had no impact on WNT signaling, though cosilencing of all three family members decreased WNT pathway activity. Finally, we characterized two moderately selective and potent small-molecule inhibitors of the CSNK1γ family. We show that these inhibitors and a CSNK1γ3 kinase-dead mutant suppressed but did not eliminate WNT-driven low-density lipoprotein receptor-related protein 6 phosphorylation and β-catenin stabilization. Our data suggest that while CSNK1γ3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway.
Topics: Casein Kinase I; Humans; Low Density Lipoprotein Receptor-Related Protein-6; Phosphorylation; Wnt Proteins; Wnt Signaling Pathway; beta Catenin
PubMed: 35487243
DOI: 10.1016/j.jbc.2022.101986 -
The Journal of Biological Chemistry Nov 2019The -encoded phosphatidate phosphatase in plays a major role in triacylglycerol synthesis and the control of phospholipid synthesis. For its catalytic function on the...
The -encoded phosphatidate phosphatase in plays a major role in triacylglycerol synthesis and the control of phospholipid synthesis. For its catalytic function on the nuclear/endoplasmic reticulum membrane, Pah1 translocates to the membrane through its phosphorylation/dephosphorylation. Pah1 phosphorylation on multiple serine/threonine residues is complex and catalyzed by diverse protein kinases. In this work, we demonstrate that Pah1 is phosphorylated by the -encoded casein kinase I (CKI), regulating Pah1 catalytic activity and phosphorylation. Phosphoamino acid analysis coupled with phosphopeptide mapping of the CKI-phosphorylated Pah1 indicated that it is phosphorylated mainly on multiple serine residues. Using site-directed mutagenesis and phosphorylation analysis of Pah1, we identified eight serine residues ( Ser-114, Ser-475, Ser-511, Ser-602, Ser-677, Ser-705, Ser-748, and Ser-774) as the target sites of CKI. Of these residues, Ser-475 and Ser-511 were specific for CKI, whereas the others were shared by casein kinase II (Ser-705), Cdc28-cyclin B (Ser-602), Pho85-Pho80 (Ser-114, Ser-602, and Ser-748), protein kinase A (Ser-667 and Ser-774), and protein kinase C (Ser-677). CKI-mediated phosphorylation of Pah1 stimulated both its phosphatidate phosphatase activity and its subsequent phosphorylation by casein kinase II. However, the CKI-mediated phosphorylation of Pah1 strongly inhibited its subsequent phosphorylation by Pho85-Pho80, protein kinase A, and protein kinase C. In a reciprocal analysis, Pah1 phosphorylation by Pho85-Pho80 inhibited subsequent phosphorylation by CKI. CKI-mediated Pah1 phosphorylation was also inhibited by a peptide containing the Pah1 residues 506-517, including the kinase-specific Ser-511 residue. These findings advance our understanding of how Pah1 catalytic activity and phosphorylation are regulated by multiple protein kinases.
Topics: Adenosine Triphosphate; Binding Sites; Biocatalysis; Casein Kinase I; Casein Kinase II; Kinetics; Mutagenesis, Site-Directed; Phosphatidate Phosphatase; Phosphorylation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Serine; Threonine
PubMed: 31645435
DOI: 10.1074/jbc.RA119.011314 -
Frontiers in Cellular and Infection... 2021Casein Kinase 1 (CK1) family members are serine/threonine protein kinases that are involved in many biological processes and highly conserved in eukaryotes from... (Review)
Review
Casein Kinase 1 (CK1) family members are serine/threonine protein kinases that are involved in many biological processes and highly conserved in eukaryotes from protozoan to humans. Even though pathogens exploit host CK1 signaling pathways to survive, the role of CK1 in infectious diseases and host/pathogen interaction is less well characterized compared to other diseases, such as cancer or neurodegenerative diseases. Here we present the current knowledge on CK1 in protozoan parasites highlighting their essential role for parasite survival and their importance for host-pathogen interactions. We also discuss how the dual requirement of CK1 family members for parasite biological processes and host subversion could be exploited to identify novel antimicrobial interventions.
Topics: Animals; Biology; Casein Kinase I; Humans; Neoplasms; Parasites; Signal Transduction
PubMed: 33869086
DOI: 10.3389/fcimb.2021.655700 -
International Journal of Molecular... Feb 2020Casein kinase I (CK1), a ubiquitous serine/threonine (Ser/Thr) protein kinase in eukaryotes, plays pivotal roles in a wide spectrum of cellular functions including... (Review)
Review
Casein kinase I (CK1), a ubiquitous serine/threonine (Ser/Thr) protein kinase in eukaryotes, plays pivotal roles in a wide spectrum of cellular functions including metabolism, cell cycle progression, developmental control and stress responses. Plant CK1 evolves a lineage expansion, resulting in a unique branch of members exclusive to the kingdom. Among them, Arabidopsis Mut9p-LIKE KINASEs (MLKs) target diverse substrates including histones and the key regulatory proteins involving in physiological processes of light signaling, circadian rhythms, phytohormone and plant defense. Deregulation of the kinase activity by mutating the enzyme or the phosphorylation sites of substrates causes developmental disorders and susceptibility to adverse environmental conditions. MLKs have evolved as a general kinase that modifies transcription factors or primary regulatory proteins in a dynamic way. Here, we summarize the current knowledge of the roles of and orthologs in several commercially important crops.
Topics: Arabidopsis Proteins; Casein Kinases; Crops, Agricultural; Plant Development; Protein Serine-Threonine Kinases; Signal Transduction
PubMed: 32106561
DOI: 10.3390/ijms21051562 -
Molecular Cell Mar 2021In our 24/7 well-lit world, it's easy to skip or delay sleep to work, study, and play. However, our circadian rhythms are not easily fooled; the consequences of jet lag... (Review)
Review
In our 24/7 well-lit world, it's easy to skip or delay sleep to work, study, and play. However, our circadian rhythms are not easily fooled; the consequences of jet lag and shift work are many and severe, including metabolic, mood, and malignant disorders. The internal clock that keeps track of time has at its heart the reversible phosphorylation of the PERIOD proteins, regulated by isoforms of casein kinase 1 (CK1). In-depth biochemical, genetic, and structural studies of these kinases, their mutants, and their splice variants have combined over the past several years to provide a robust understanding of how the core clock is regulated by a phosphoswitch whereby phosphorylation of a stabilizing site on PER blocks phosphorylation of a distant phosphodegron. The recent structure of a circadian mutant form of CK1 implicates an internal activation loop switch that regulates this phosphoswitch and points to new approaches to regulation of the clock.
Topics: Animals; Casein Kinase I; Circadian Clocks; Circadian Rhythm; Humans; Period Circadian Proteins; Phosphorylation
PubMed: 33545069
DOI: 10.1016/j.molcel.2021.01.006