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1.
bioRxiv ; 2024 May 17.
Article in English | MEDLINE | ID: mdl-38798407

ABSTRACT

Mutations in human CILK1 (ciliogenesis associated kinase 1) are linked to ciliopathies and epilepsy. Homozygous point and nonsense mutations that extinguish kinase activity impair primary cilia function, whereas mutations outside the kinase domain are not well understood. Here, we produced a knock-in mouse equivalent of the human CILK1 A615T variant identified in juvenile myoclonic epilepsy (JME). This residue is in the C-terminal region of CILK1 separate from the kinase domain. Mouse embryo fibroblasts (MEF) with either heterozygous or homozygous A612T mutant alleles exhibited a higher ciliation rate, shorter individual cilia and up-regulation of ciliary Hedgehog signaling. Thus, a single A612T mutant allele was sufficient to impair primary cilia and ciliary signaling in MEFs. Gene expression profiles of wild type versus mutant MEFs revealed profound changes in cilia-related molecular functions and biological processes. CILK1 A615T mutant protein was not increased to the same level as the wild type protein when co-expressed with scaffold protein KATNIP (katanin-interacting protein). Our data show that KATNIP regulation of a JME-associated single residue variant of CILK1 is compromised and this impairs the maintenance of primary cilia and Hedgehog signaling.

2.
Mol Cell Biol ; 43(9): 472-480, 2023.
Article in English | MEDLINE | ID: mdl-37665596

ABSTRACT

The primary cilium functions as a cellular sensory organelle and signaling antenna that detects and transduces extracellular signals. Mutations in the human gene CILK1 (ciliogenesis associated kinase 1) cause abnormal cilia elongation and faulty Hedgehog signaling, associated with developmental disorders and epilepsy. CILK1 is a protein kinase that requires dual phosphorylation of its TDY motif for activation and its extended C-terminal intrinsically disordered region (IDR) mediates targeting to the basal body and substrate recognition. Proteomics previously identified katanin-interacting protein (KATNIP), also known as KIAA0556, as a CILK1 interacting partner. In this study we discovered that CILK1 colocalizes with KATNIP at the basal body and the CILK1 IDR is sufficient to mediate binding to KATNIP. Deletion analysis of KATNIP shows one of three domains of unknown function (DUF) is required for association with CILK1. KATNIP binding with CILK1 drastically elevated CILK1 protein levels and TDY phosphorylation in cells. This resulted in a profound increase in phosphorylation of known CILK1 substrates and suppression of cilia length. Thus, KATNIP functions as a regulatory subunit of CILK1 that potentiates its actions. This advances our understanding of the molecular basis of control of primary cilia.


Subject(s)
Cilia , Humans , Cilia/metabolism , Hedgehog Proteins , Katanin , Phosphorylation , Signal Transduction
3.
IUBMB Life ; 75(4): 328-336, 2023 04.
Article in English | MEDLINE | ID: mdl-36239169

ABSTRACT

Glycogen is a polymerized form of glucose that serves as an energy reserve in all types of organisms. In animals glycogen synthesis and degradation, especially in liver and skeletal muscle, are regulated by hormonal and physiological signals that reciprocally control the opposing activities of glycogen synthase and glycogen phosphorylase. These enzymes are under allosteric control by binding of metabolites (e.g., ATP, AMP, G6P) and covalent control by reversible phosphorylation by kinase and phosphatase all assembled together on glycogen. More than 50 years ago Edmond Fischer and colleagues showed "flash activation" of phosphorylase in glycogen particles. This involved transient and extensive inhibition of protein phosphatase but even today the phenomenon is not understood. Phosphatase regulation is known to rely on regulatory subunits including glycogen binding subunits that serve as scaffolds, binding catalytic subunit, glycogen, and substrates. This tribute article to Edmond Fischer highlights his thoughts and ideas about the transient inhibition of phosphorylase phosphatase during flash activation of phosphorylase and speculates that phosphatase regulation in glycogen particles might involve a/b hybrids of phosphorylase.


Subject(s)
Phosphoprotein Phosphatases , Phosphorylase Phosphatase , Animals , Phosphoprotein Phosphatases/metabolism , Glycogen , Glycogen Phosphorylase/genetics , Glycogen Phosphorylase/metabolism , Phosphorylases/genetics , Phosphorylases/metabolism , Muscle, Skeletal/metabolism , Liver/metabolism
4.
Elife ; 112022 11 23.
Article in English | MEDLINE | ID: mdl-36416764

ABSTRACT

Genome-wide association studies (GWASs) for bone mineral density (BMD) in humans have identified over 1100 associations to date. However, identifying causal genes implicated by such studies has been challenging. Recent advances in the development of transcriptome reference datasets and computational approaches such as transcriptome-wide association studies (TWASs) and expression quantitative trait loci (eQTL) colocalization have proven to be informative in identifying putatively causal genes underlying GWAS associations. Here, we used TWAS/eQTL colocalization in conjunction with transcriptomic data from the Genotype-Tissue Expression (GTEx) project to identify potentially causal genes for the largest BMD GWAS performed to date. Using this approach, we identified 512 genes as significant using both TWAS and eQTL colocalization. This set of genes was enriched for regulators of BMD and members of bone relevant biological processes. To investigate the significance of our findings, we selected PPP6R3, the gene with the strongest support from our analysis which was not previously implicated in the regulation of BMD, for further investigation. We observed that Ppp6r3 deletion in mice decreased BMD. In this work, we provide an updated resource of putatively causal BMD genes and demonstrate that PPP6R3 is a putatively causal BMD GWAS gene. These data increase our understanding of the genetics of BMD and provide further evidence for the utility of combined TWAS/colocalization approaches in untangling the genetics of complex traits.


Subject(s)
Genome-Wide Association Study , Quantitative Trait Loci , Humans , Mice , Animals , Transcriptome , Bone Density/genetics , Genetic Predisposition to Disease
5.
Int J Mol Sci ; 23(15)2022 Jul 23.
Article in English | MEDLINE | ID: mdl-35897693

ABSTRACT

The primary cilium provides cell sensory and signaling functions. Cilia structure and function are regulated by ciliogenesis-associated kinase 1 (CILK1). Ciliopathies caused by CILK1 mutations show longer cilia and abnormal Hedgehog signaling. Our study aimed to identify small molecular inhibitors of CILK1 that would enable pharmacological modulation of primary cilia. A previous screen of a chemical library for interactions with protein kinases revealed that Alvocidib has a picomolar binding affinity for CILK1. In this study, we show that Alvocidib potently inhibits CILK1 (IC50 = 20 nM), exhibits selectivity for inhibition of CILK1 over cyclin-dependent kinases 2/4/6 at low nanomolar concentrations, and induces CILK1-dependent cilia elongation. Our results support the use of Alvocidib to potently and selectively inhibit CILK1 to modulate primary cilia.


Subject(s)
Cilia , Ciliopathies , Cilia/metabolism , Ciliopathies/metabolism , Flavonoids/metabolism , Hedgehog Proteins/metabolism , Humans , Piperidines
6.
Radiol Imaging Cancer ; 4(4): e210098, 2022 07.
Article in English | MEDLINE | ID: mdl-35838531

ABSTRACT

Animal models play a crucial role in developing and testing new therapies for hepatocellular carcinoma (HCC), providing preclinical evidence prior to exploring human safety and efficacy outcomes. The interventional radiologist must weigh the advantages and disadvantages of various animal models available when testing a new local-regional therapy. This review highlights the currently available animal models for testing local-regional therapies for HCC and details the importance of considering animal genetics, tumor biology, and molecular mechanisms when ultimately choosing an animal model. Keywords: Animal Studies, Interventional-Vascular, Molecular Imaging-Clinical Translation, Molecular Imaging-Cancer, Chemoembolization, Liver © RSNA, 2022.


Subject(s)
Carcinoma, Hepatocellular , Chemoembolization, Therapeutic , Liver Neoplasms , Animals , Carcinoma, Hepatocellular/diagnostic imaging , Carcinoma, Hepatocellular/therapy , Humans , Liver Neoplasms/diagnostic imaging , Liver Neoplasms/therapy , Models, Animal , Tomography, X-Ray Computed
7.
Circ Res ; 130(1): 96-111, 2022 01 07.
Article in English | MEDLINE | ID: mdl-34794320

ABSTRACT

BACKGROUND: How signals from activated angiotensin type-2 receptors (AT2R) mediate inhibition of sodium ion (Na+) reabsorption in renal proximal tubule cells is currently unknown. Protein phosphatases including PP2A (protein phosphatase 2A) have been implicated in AT2R signaling in tissues other than kidney. We investigated whether inhibition of protein phosphatase PP2A reduced AT2R-mediated natriuresis and evaluated changes in PP2A activity and localization after renal AT2R activation in normal 4- and 10-week-old control Wistar-Kyoto rats and 4-week-old prehypertensive and 10-week-old hypertensive spontaneously hypertensive rats. METHODS AND RESULTS: In Wistar-Kyoto rats, direct renal interstitial administration of selective AT2R nonpeptide agonist Compound-21 (C-21) increased renal interstitial cyclic GMP (cGMP) levels, urine Na+ excretion, and simultaneously increased PP2A activity ≈2-fold in homogenates of renal cortical tubules. The cyclic GMP and natriuretic responses were abolished by concurrent renal interstitial administration of protein phosphatase inhibitor calyculin A. In renal proximal tubule cells in response to C-21, PP2A subunits A, B55α and C, but not B56γ, were recruited to apical plasma membranes together with AT2Rs. Calyculin A treatment abolished C-21-induced translocation of both AT2R and PP2A regulatory subunit B55α to apical plasma membranes. Immunoprecipitation of AT2R solubilized from renal cortical homogenates demonstrated physical association of AT2R with PP2A A, B55α, and C but not B56γ subunits. In contrast, in spontaneously hypertensive rats, administration of C-21 did not alter urine Na+ excretion or PP2A activity and failed to translocate AT2Rs and PP2A subunits to apical plasma membranes. CONCLUSIONS: In renal proximal tubule cells of Wistar-Kyoto rats, PP2A is activated and PP2A subunits AB55αC are recruited to C-21-activated AT2Rs during induction of natriuresis. This response is defective in prehypertensive and hypertensive spontaneously hypertensive rats, presenting a potential novel therapeutic target for treating renal Na+ retention and hypertension.


Subject(s)
Kidney/metabolism , Natriuresis , Protein Phosphatase 2/metabolism , Receptor, Angiotensin, Type 2/metabolism , Animals , Cells, Cultured , Cyclic GMP/metabolism , Female , Rats , Rats, Wistar , Sodium/metabolism
8.
Nat Commun ; 12(1): 6207, 2021 10 27.
Article in English | MEDLINE | ID: mdl-34707113

ABSTRACT

Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), produced by cyclic GMP-AMP synthase (cGAS), stimulates the production of type I interferons (IFN). Here we show that cGAMP activates DNA damage response (DDR) signaling independently of its canonical IFN pathways. Loss of cGAS dampens DDR signaling induced by genotoxic insults. Mechanistically, cGAS activates DDR in a STING-TBK1-dependent manner, wherein TBK1 stimulates the autophosphorylation of the DDR kinase ATM, with the consequent activation of the CHK2-p53-p21 signal transduction pathway and the induction of G1 cell cycle arrest. Despite its stimulatory activity on ATM, cGAMP suppresses homology-directed repair (HDR) through the inhibition of polyADP-ribosylation (PARylation), in which cGAMP reduces cellular levels of NAD+; meanwhile, restoring NAD+ levels abrogates cGAMP-mediated suppression of PARylation and HDR. Finally, we show that cGAMP also activates DDR signaling in invertebrate species lacking IFN (Crassostrea virginica and Nematostella vectensis), suggesting that the genome surveillance mechanism of cGAS predates metazoan interferon-based immunity.


Subject(s)
DNA Damage , Nucleotides, Cyclic/metabolism , Signal Transduction , Animals , Ataxia Telangiectasia Mutated Proteins/metabolism , Crassostrea/genetics , Crassostrea/metabolism , G1 Phase Cell Cycle Checkpoints , Humans , Immunity, Innate , Interferon Type I/metabolism , Membrane Proteins/metabolism , Mice , Nucleotidyltransferases/metabolism , Phosphorylation , Poly ADP Ribosylation , Protein Serine-Threonine Kinases/metabolism , Recombinational DNA Repair , Sea Anemones/genetics , Sea Anemones/metabolism
9.
Clin Sci (Lond) ; 135(13): 1545-1556, 2021 07 16.
Article in English | MEDLINE | ID: mdl-34192314

ABSTRACT

Tumor initiation is driven by oncogenes that activate signaling networks for cell proliferation and survival involving protein phosphorylation. Protein kinases in these pathways have proven to be effective targets for pharmaceutical inhibitors that have progressed to the clinic to treat various cancers. Here, we offer a narrative about the development of small molecule modulators of the protein Ser/Thr phosphatase 2A (PP2A) to reduce the activation of cell proliferation and survival pathways. These novel drugs promote the assembly of select heterotrimeric forms of PP2A that act to limit cell proliferation. We discuss the potential for the near-term translation of this approach to the clinic for cancer and other human diseases.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Design , Enzyme Inhibitors/pharmacology , Neoplasms/drug therapy , Protein Phosphatase 2/antagonists & inhibitors , Allosteric Regulation , Animals , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Enzyme Inhibitors/chemistry , Humans , Molecular Targeted Therapy , Neoplasms/enzymology , Neoplasms/pathology , Phosphorylation , Protein Conformation , Protein Phosphatase 2/chemistry , Protein Phosphatase 2/metabolism , Signal Transduction , Structure-Activity Relationship
10.
Dev Dyn ; 250(2): 263-273, 2021 02.
Article in English | MEDLINE | ID: mdl-32935890

ABSTRACT

BACKGROUND: Kinesin family member 3A (KIF3A) is a molecular motor protein in the heterotrimeric kinesin-2 complex that drives anterograde intraflagellar transport. This process plays a pivotal role in both biogenesis and maintenance of the primary cilium that supports tissue development. Ciliogenesis associated kinase 1 (CILK1) phosphorylates human KIF3A at Thr672. CILK1 loss of function causes ciliopathies that manifest profound and multiplex developmental defects, including hydrocephalus, polydactyly, shortened and hypoplastic bones and alveoli airspace deficiency, leading to perinatal lethality. Prior studies have raised the hypothesis that CILK1 phosphorylation of KIF3A is critical for its regulation of organ development. RESULTS: We produced a mouse model with phosphorylation site Thr674 in mouse Kif3a mutated to Ala. Kif3a T674A homozygotes are viable and exhibit no skeletal and brain abnormalities, and only mildly reduced airspace in alveoli. Mouse embryonic fibroblasts carrying Kif3a T674A mutation show a normal rate of ciliation and a moderate increase in cilia length. CONCLUSION: These results indicate that eliminating Kif3a Thr674 phosphorylation by Cilk1 is insufficient to reproduce the severe developmental defects in ciliopathies caused by Cilk1 loss of function. This suggests KIF3A-Thr672 phosphorylation by CILK1 is not essential for tissue development and other substrates are involved in CILK1 ciliopathies.


Subject(s)
Cilia , Ciliopathies/genetics , Kinesins/physiology , Loss of Function Mutation , Protein Serine-Threonine Kinases/genetics , Animals , Disease Models, Animal , Female , Gene Knock-In Techniques , Male , Mice, Transgenic , Phenotype
11.
Cell ; 181(3): 688-701.e16, 2020 04 30.
Article in English | MEDLINE | ID: mdl-32315618

ABSTRACT

Impairment of protein phosphatases, including the family of serine/threonine phosphatases designated PP2A, is essential for the pathogenesis of many diseases, including cancer. The ability of PP2A to dephosphorylate hundreds of proteins is regulated by over 40 specificity-determining regulatory "B" subunits that compete for assembly and activation of heterogeneous PP2A heterotrimers. Here, we reveal how a small molecule, DT-061, specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate selective substrates, such as its well-known oncogenic target, c-Myc. Our 3.6 Å structure identifies molecular interactions between DT-061 and all three PP2A subunits that prevent dissociation of the active enzyme and highlight inherent mechanisms of PP2A complex assembly. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets.


Subject(s)
Protein Phosphatase 2/metabolism , Amino Acid Sequence , Animals , Cell Line, Tumor , Enzyme Activators/metabolism , HEK293 Cells , Heterografts , Humans , Male , Mice , Mice, Nude , Models, Molecular , Multiprotein Complexes/metabolism , Protein Phosphatase 2/chemistry , Protein Subunits
12.
Cells ; 9(3)2020 03 12.
Article in English | MEDLINE | ID: mdl-32178256

ABSTRACT

Ciliopathies are a group of human genetic disorders associated with mutations that give rise to the dysfunction of primary cilia. Ciliogenesis-associated kinase 1 (CILK1), formerly known as intestinal cell kinase (ICK), is a conserved serine and threonine kinase that restricts primary (non-motile) cilia formation and length. Mutations in CILK1 are associated with ciliopathies and are also linked to juvenile myoclonic epilepsy (JME). However, the effects of the JME-related mutations in CILK1 on kinase activity and CILK1 function are unknown. Here, we report that JME pathogenic mutations in the CILK1 N-terminal kinase domain abolish kinase activity, evidenced by the loss of phosphorylation of kinesin family member 3A (KIF3A) at Thr672, while JME mutations in the C-terminal non-catalytic domain (CTD) have little effect on KIF3A phosphorylation. Although CILK1 variants in the CTD retain catalytic activity, they nonetheless lose the ability to restrict cilia length and also gain function in promoting ciliogenesis. We show that wild type CILK1 predominantly localizes to the base of the primary cilium; in contrast, JME variants of CILK1 are distributed along the entire axoneme of the primary cilium. These results demonstrate that JME pathogenic mutations perturb CILK1 function and intracellular localization. These CILK1 variants affect the primary cilium, independent of CILK1 phosphorylation of KIF3A. Our findings suggest that CILK1 mutations linked to JME result in alterations of primary cilia formation and homeostasis.


Subject(s)
Myoclonic Epilepsy, Juvenile/genetics , Protein Serine-Threonine Kinases/genetics , Animals , Cilia/pathology , HEK293 Cells , Humans , Mice , Mutation , Myoclonic Epilepsy, Juvenile/enzymology , Myoclonic Epilepsy, Juvenile/pathology , NIH 3T3 Cells , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Transfection
13.
Biochem J ; 477(2): 431-444, 2020 01 31.
Article in English | MEDLINE | ID: mdl-31904830

ABSTRACT

Protein Ser/Thr phosphatase-6 (PP6) regulates pathways for activation of NF-kB, YAP1 and Aurora A kinase (AURKA). PP6 is a heterotrimer comprised of a catalytic subunit, one of three different SAPS subunits and one of three different ankyrin-repeat ANKRD subunits. Here, we show FLAG-PP6C expressed in cells preferentially binds endogenous SAPS3, and the complex is active with the chemical substrate DiFMUP. SAPS3 has multiple acidic sequence motifs recognized by protein kinase CK2 (CK2) and SAPS3 is phosphorylated by purified CK2, without affecting its associated PP6 phosphatase activity. However, HA3-SAPS3-PP6 phosphatase activity using pT288 AURKA as substrate is significantly increased by phosphorylation with CK2. The substitution of Ala in nine putative phosphorylation sites in SAPS3 was required to prevent CK2 activation of the phosphatase. Different CK2 chemical inhibitors equally increased phosphorylation of endogenous AURKA in living cells, consistent with reduction in PP6 activity. CRISPR/Cas9 deletion or siRNA knockdown of SAPS3 resulted in highly activated endogenous AURKA, and a high proportion of cells with abnormal nuclei. Activation of PP6 by CK2 can form a feedback loop with bistable changes in substrates.


Subject(s)
Aurora Kinase A/genetics , Casein Kinase II/chemistry , Phosphoprotein Phosphatases/genetics , Alanine/genetics , Amino Acid Substitution/genetics , Aurora Kinase A/chemistry , CRISPR-Cas Systems/genetics , Casein Kinase II/genetics , Catalytic Domain/genetics , Enzyme Inhibitors/pharmacology , HeLa Cells , Humans , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/chemistry , Phosphorylation/genetics , Protein Binding/drug effects , RNA, Small Interfering/genetics , Substrate Specificity/drug effects
14.
Oncogene ; 39(3): 703-717, 2020 01.
Article in English | MEDLINE | ID: mdl-31541192

ABSTRACT

The serine/threonine Protein Phosphatase 2A (PP2A) functions as a tumor suppressor by negatively regulating multiple oncogenic signaling pathways. The canonical PP2A holoenzyme comprises a scaffolding subunit (PP2A Aα/ß), which serves as the platform for binding of both the catalytic C subunit and one regulatory B subunit. Somatic heterozygous missense mutations in PPP2R1A, the gene encoding the PP2A Aα scaffolding subunit, have been identified across multiple cancer types, but the effects of the most commonly mutated residue, Arg-183, on PP2A function have yet to be fully elucidated. In this study, we used a series of cellular and in vivo models and discovered that the most frequent Aα R183W mutation formed alternative holoenzymes by binding of different PP2A regulatory subunits compared with wild-type Aα, suggesting a rededication of PP2A functions. Unlike wild-type Aα, which suppressed tumorigenesis, the R183W mutant failed to suppress tumor growth in vivo through activation of the MAPK pathway in RAS-mutant transformed cells. Furthermore, cells expressing R183W were less sensitive to MEK inhibitors. Taken together, our results demonstrate that the R183W mutation in PP2A Aα scaffold abrogates the tumor suppressive actions of PP2A, thereby potentiating oncogenic signaling and reducing drug sensitivity of RAS-mutant cells.


Subject(s)
Drug Resistance, Neoplasm/genetics , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Phosphatase 2/genetics , Recombinant Proteins/genetics , Amino Acid Substitution , Arginine/genetics , Calmodulin-Binding Proteins/metabolism , Carcinogenesis/drug effects , Carcinogenesis/genetics , Cell Line, Tumor , Humans , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Membrane Proteins/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Mutation , Neoplasms/genetics , Nerve Tissue Proteins/metabolism , Protein Kinase Inhibitors/therapeutic use , Protein Phosphatase 2/isolation & purification , Recombinant Proteins/isolation & purification , Transfection , Tyrosine/genetics , Xenograft Model Antitumor Assays
15.
DNA Repair (Amst) ; 85: 102737, 2020 01.
Article in English | MEDLINE | ID: mdl-31751917

ABSTRACT

Cellular responses to DNA damage include activation of DNA-dependent protein kinase (DNA-PK) through, among others, the serine/threonine protein phosphatase 6 (PP6). We previously showed that recognition of DNA-PKcs is mediated by the SAPS1 PP6 regulatory subunit. Here, we report and characterize a SAPS1 null mouse and investigate the effects of deletion on DNA damage signaling and repair. Strikingly, neither SAPS1-null animals nor cells derived from them show gross defects, unless subjected to DNA damage by radiation or chemical agents. The overall survival of SAPS1-null animals following whole body irradiation is significantly shortened as compared to wild-type mice, and the clonogenic survival of null cells subjected to ionizing radiation is reduced. The dephosphorylation of DNA damage/repair markers, such as γH2AX, p53 and Kap1, is diminished in SAPS1-null cells as compared to wild-type controls. Our results demonstrate that loss of SAPS1 confers sensitivity to DNA damage and confirms previously reported cellular phenotypes of SAPS1 knock-down in human glioma cells. The results support a role for PP6 regulatory subunit SAPS1 in DNA damage responses, and offer a novel target for sensitization to enhance current tumor therapies, with a potential for limited deleterious side effects.


Subject(s)
DNA-Activated Protein Kinase/genetics , Loss of Function Mutation , Phosphoprotein Phosphatases/metabolism , Whole-Body Irradiation/adverse effects , Animals , Cells, Cultured , DNA Damage , DNA Repair , Histones/metabolism , Mice , Phosphoprotein Phosphatases/genetics , Phosphorylation , Tripartite Motif-Containing Protein 28/metabolism , Tumor Suppressor Protein p53/metabolism
16.
Mol Cancer Ther ; 19(4): 1008-1017, 2020 04.
Article in English | MEDLINE | ID: mdl-31848297

ABSTRACT

Hypoxia is a major factor in tumor progression and resistance to therapies, which involves elevated levels of the transcription factor HIF1α. Here, we report that prostate tumor xenografts express high levels of HIF1α and show greatly enhanced growth in response to knockdown of the E3 ligase CHIP (C-terminus of Hsp70-interacting protein). In multiple human prostate cancer cell lines under hypoxia, taxol treatment induces the degradation of HIF1α, and this response is abrogated by knockdown of CHIP, but not by E3 ligase VHL or RACK1. HIF1α degradation is accompanied by loss of function, evidenced by reduced expression of HIF1α-dependent genes. CHIP-dependent HIF1α degradation also occurs in cells arrested in mitosis by nocodazole instead of taxol. Mitotic kinase Aurora B activity is required for taxol-induced HIF1α degradation. Purified Aurora B directly phosphorylates HIF1α at multiple sites, and these modifications enhance its polyubiquitination by CHIP in a purified reconstituted system. Our results show how activation of Aurora B promotes CHIP-dependent degradation of HIF1α in prostate cancer cells. This new knowledge may affect the use of mitotic kinase inhibitors and open new approaches for treatment of hypoxic prostate tumors.


Subject(s)
Aurora Kinase B/metabolism , Biomarkers, Tumor/metabolism , Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Prostatic Neoplasms/pathology , Proteolysis , Ubiquitin-Protein Ligases/metabolism , Apoptosis , Aurora Kinase B/genetics , Biomarkers, Tumor/genetics , Cell Proliferation , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Paclitaxel/pharmacology , Phosphorylation , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Tumor Cells, Cultured , Ubiquitin-Protein Ligases/genetics , Ubiquitination
17.
FEBS Lett ; 593(21): 2990-3002, 2019 11.
Article in English | MEDLINE | ID: mdl-31506943

ABSTRACT

Ciliogenesis associated kinase 1 (CILK1) was previously known as intestinal cell kinase because it was cloned from that origin. However, CILK1 is now recognized as a widely expressed and highly conserved serine/threonine protein kinase. Mutations in the human CILK1 gene have been associated with ciliopathies, a group of human genetic disorders with defects in the primary cilium. In mice, both Cilk1 knock-out and Cilk1 knock-in mutations have recapitulated human ciliopathies. Thus, CILK1 has a fundamental role in the function of the cilium. Several candidate substrates have been proposed for CILK1 and the challenge is to relate these to the mutant phenotypes. In this review, we summarize what is known about CILK1 functions and targets, and discuss gaps in current knowledge that motivate further experimentation to fully understand the role of CILK1 in organ development in humans.


Subject(s)
Cilia/metabolism , Ciliopathies/genetics , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Animals , Ciliopathies/metabolism , Disease Models, Animal , Gene Expression Regulation , Humans , Mice , Mutation , Phosphorylation , Protein Serine-Threonine Kinases/genetics
18.
Cells ; 8(7)2019 07 04.
Article in English | MEDLINE | ID: mdl-31277411

ABSTRACT

Loss-of-function mutations in the human ICK (intestinal cell kinase) gene cause dysfunctional primary cilia and perinatal lethality which are associated with human ciliopathies. The enzyme that we herein call CAPK (ciliopathy-associated protein kinase) is a serine/threonine protein kinase that has a highly conserved MAPK-like N-terminal catalytic domain and an unstructured C-terminal domain (CTD) whose functions are completely unknown. In this study, we demonstrate that truncation of the CTD impairs the ability of CAPK to interact with and phosphorylate its substrate, kinesin family member 3A (KIF3A). We also find that deletion of the CTD of CAPK compromises both localization to the primary cilium and negative regulation of ciliogenesis. Thus, CAPK substrate recognition, ciliary targeting, and ciliary function depend on the non-catalytic CTD of the protein which is predicted to be intrinsically disordered.


Subject(s)
Cilia/physiology , Kinesins/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , HEK293 Cells , Humans , Mice , NIH 3T3 Cells , Phosphorylation/genetics , Protein Domains/genetics , Protein Serine-Threonine Kinases/genetics
19.
FASEB J ; 33(9): 9945-9958, 2019 09.
Article in English | MEDLINE | ID: mdl-31157564

ABSTRACT

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder causing renal failure. Mutations of polycystic kidney disease 1 (PKD1) account for most ADPKD cases. Defective ciliary localization of polycystin-1 (PC1), a large integral membrane protein encoded by PKD1, underlies the pathogenesis of a subgroup of patients with ADPKD. However, the mechanisms by which PC1 and other ciliary proteins traffic to the primary cilium remain poorly understood. A ciliary targeting sequence (CTS) that resides in ciliary receptors is considered to function in the process. It has been reported that the VxP motif in the intracellular C-terminal tail of PC1 functions as a CTS in an ADP ribosylation factor 4 (Arf4)/ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1)-dependent manner. However, other recent studies have revealed that this motif is dispensable for PC1 trafficking to cilia. In this study, we identified a novel CTS consisting of 8 residues (RHKVRFEG) in the PC1 C tail. We found that this motif is sufficient to bind protein phosphatase 1 (PP1)α, a ubiquitously expressed phosphatase in the phosphoprotein phosphatase (PPP) family. Mutations in this CTS motif disrupt binding with PP1α and impair ciliary localization of PC1. Additionally, short hairpin RNA-mediated knockdown of PP1α results in reduced ciliary localization of PC1 and elongated cilia, suggesting a role for PP1α in the regulation of ciliary structure and function.-Luo, C., Wu, M., Su, X., Yu, F., Brautigan, D. L., Chen, J., Zhou, J. Protein phosphatase 1α interacts with a novel ciliary targeting sequence of polycystin-1 and regulates polycystin-1 trafficking.


Subject(s)
Protein Phosphatase 1/metabolism , TRPP Cation Channels/metabolism , Alanine , Amino Acid Sequence , Animals , Cell Line , Mice , Mice, Knockout , Mutagenesis , Protein Phosphatase 1/genetics , Protein Transport , TRPP Cation Channels/genetics
20.
J Mol Cell Cardiol ; 127: 204-214, 2019 02.
Article in English | MEDLINE | ID: mdl-30597148

ABSTRACT

Over 5 million people in the United States suffer from heart failure, due to the limited ability to regenerate functional cardiac tissue. One potential therapeutic strategy is to enhance proliferation of resident cardiomyocytes. However, phenotypic screening for therapeutic agents is challenged by the limited ability of conventional markers to discriminate between cardiomyocyte proliferation and endoreplication (e.g. polyploidy and multinucleation). Here, we developed a novel assay that combines automated live-cell microscopy and image processing algorithms to discriminate between proliferation and endoreplication by quantifying changes in the number of nuclei, changes in the number of cells, binucleation, and nuclear DNA content. We applied this assay to further prioritize hits from a primary screen for DNA synthesis, identifying 30 compounds that enhance proliferation of human induced pluripotent stem cell-derived cardiomyocytes. Among the most active compounds from the phenotypic screen are clinically approved L-type calcium channel blockers from multiple chemical classes whose activities were confirmed across different sources of human induced pluripotent stem cell-derived cardiomyocytes. Identification of compounds that stimulate human cardiomyocyte proliferation may provide new therapeutic strategies for heart failure.


Subject(s)
Calcium Channels, L-Type/metabolism , Induced Pluripotent Stem Cells/cytology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Cell Proliferation , DNA/biosynthesis , Humans , Image Processing, Computer-Assisted , Phenotype , Ploidies
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