scholarly journals Ciliopathy-Associated Protein Kinase ICK Requires Its Non-Catalytic Carboxyl-Terminal Domain for Regulation of Ciliogenesis

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 677 ◽  
Author(s):  
Yoon Seon Oh ◽  
Eric J. Wang ◽  
Casey D. Gailey ◽  
David L. Brautigan ◽  
Benjamin L. Allen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Primary Cilia ◽  
Catalytic Domain ◽  
Intestinal Cell ◽  
Loss Of Function ◽  
Kinase Gene ◽  
Intrinsically Disordered ◽  
Terminal Domain ◽  
Perinatal Lethality ◽  
Carboxyl Terminal

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.

scholarly journals The Carboxyl-Terminal Domain of the Protein Kinase Fused Can Function as a Dominant Inhibitor of Hedgehog Signaling

2002 ◽  
Vol 22 (5) ◽  
pp. 1555-1566 ◽  
Author(s):  
Manuel Ascano ◽  
Kent E. Nybakken ◽  
Janek Sosinski ◽  
Melanie A. Stegman ◽  
David J. Robbins
Keyword(s):  
Protein Kinase ◽  
Critical Role ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Signaling Complex ◽  
Terminal Domain ◽  
Cell Extracts ◽  
Carboxyl Terminal Domain ◽  
Hh Signaling ◽  
Carboxyl Terminal

ABSTRACT The secreted protein hedgehog (Hh) plays a critical role in the developmental patterning of multiple tissues. In Drosophila melanogaster, a cytosolic multiprotein signaling complex appears necessary for Hh signaling. Genes that encode components of this Hh signaling complex (HSC) were originally identified and characterized based on their genetic interactions with hh, as well as with each other. It is only in recent years that the mechanistic functions of these components have begun to be unraveled. Here, we have investigated the relationship between two components of the HSC, the serine/threonine protein kinase Fused (Fu) and the kinesin-related protein Costal2 (Cos2). We have reconstituted a Fu/Cos2 complex in vitro and shown that Fu is able to directly associate with Cos2, forming a complex whose molecular size is similar to a previously described complex found in Drosophila cell extracts. We have also determined that the carboxyl-terminal domain of Fu is necessary and sufficient for the direct binding of Fu to Cos2. To validate the physiological relevance of this interaction, we overexpressed the carboxyl-terminal domain of Fu in wild-type flies. These flies exhibit a phenotype similar to that seen in fu mutants and consistent with an hh loss-of-function phenotype. We conclude that the carboxyl-terminal domain of Fu can function in a dominant negative manner, by preventing endogenous Fu from binding to Cos2. Thus, we provide the first evidence that Hh signaling can be compromised by targeting the HSC for disruption.

scholarly journals Activation of a Nuclear Cdc2-Related Kinase within a Mitogen-Activated Protein Kinase-Like TDY Motif by Autophosphorylation and Cyclin-Dependent Protein Kinase-Activating Kinase

2005 ◽  
Vol 25 (14) ◽  
pp. 6047-6064 ◽  
Author(s):  
Zheng Fu ◽  
Melanie J. Schroeder ◽  
Jeffrey Shabanowitz ◽  
Philipp Kaldis ◽  
Kasumi Togawa ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Mitogen Activated Protein Kinase ◽  
Intestinal Cell ◽  
Dependent Protein Kinase ◽  
Mitogen Activated Protein ◽  
Dependent Protein

ABSTRACT Male germ cell-associated kinase (MAK) and intestinal cell kinase (ICK) are nuclear Cdc2-related kinases with nearly identical N-terminal catalytic domains and more divergent C-terminal noncatalytic domains. The catalytic domain is also related to mitogen-activated protein kinases (MAPKs) and contains a corresponding TDY motif. Nuclear localization of ICK requires subdomain XI and interactions of the conserved Arg-272, but not kinase activity or, surprisingly, any of the noncatalytic domain. Further, nuclear localization of ICK is required for its activation. ICK is activated by dual phosphorylation of the TDY motif. Phosphorylation of Tyr-159 in the TDY motif requires ICK autokinase activity but confers only basal kinase activity. Full activation requires additional phosphorylation of Thr-157 in the TDY motif. Coexpression of ICK with constitutively active MEK1 or MEK5 fails to increase ICK phosphorylation or activity, suggesting that MEKs are not involved. ICK and MAK are related to Ime2p in budding yeast, and cyclin-dependent protein kinase-activating kinase Cak1p has been placed genetically upstream of Ime2p. Recombinant Cak1p phosphorylates Thr-157 in the TDY motif of recombinant ICK and activates its activity in vitro. Coexpression of ICK with wild-type CAK1 but not kinase-inactive CAK1 in cells also increases ICK phosphorylation and activity. Our studies establish ICK as the prototype for a new group of MAPK-like kinases requiring dual phosphorylation at TDY motifs.

Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase II

1988 ◽  
Vol 8 (1) ◽  
pp. 330-339 ◽  
Author(s):  
M S Bartolomei ◽  
N F Halden ◽  
C R Cullen ◽  
J L Corden
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Consensus Sequence ◽  
Loss Of Function ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal ◽  
Substitution Mutations ◽  
Mutant Genes ◽  
Alpha Amanitin

The carboxyl-terminal domain (CTD) of the mouse RNA polymerase II largest subunit consists of 52 repeats of a seven-amino-acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A genetic approach was used to determine whether the CTD plays an essential role in RNA polymerase function. Deletion, insertion, and substitution mutations were created in the repetitive region of an alpha-amanitin-resistant largest-subunit gene. The effects of these mutations on RNA polymerase II activity were assayed by measuring the ability of mutant genes to confer alpha-amanitin resistance after transfection of susceptible rodent cells. Mutations that resulted in CTDs containing between 36 and 78 repeats had no effect on the transfer of alpha-amanitin resistance, whereas mutations with 25 or fewer repeats were inactive in this assay. Mutations that contained 29, 31, or 32 repeats had an intermediate effect; the number of alpha-amanitin-resistant colonies was lower and the colonies obtained were smaller, indicating that the mutant RNA polymerase II was defective. In addition, not all of the heptameric repeats were functionally equivalent in that repeats that diverged in up to three amino acids from the consensus sequence could not substitute for the conserved heptamer repeats. We concluded that the CTD is essential for RNA polymerase II activity, since substantial mutations in this region result in loss of function.

scholarly journals Dictyostelium discoideum has a single diacylglycerol kinase gene with similarity to mammalian θ isoforms

2002 ◽  
Vol 368 (3) ◽  
pp. 809-815 ◽  
Author(s):  
Marc A. de la ROCHE ◽  
Janet L. SMITH ◽  
Maribel RICO ◽  
Silvia CARRASCO ◽  
Isabel MERIDA ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dictyostelium Discoideum ◽  
Heavy Chain ◽  
Catalytic Domain ◽  
Single Gene ◽  
Myosin Ii ◽  
Terminal Segment ◽  
Gene Encoding ◽  
Kinase Gene ◽  
Myosin Heavy Chain Protein

Diacylglycerol kinases (DGKs) phosphorylate the neutral lipid diacylglycerol (DG) to produce phosphatidic acid (PA). In mammalian systems DGKs are a complex family of at least nine isoforms that are thought to participate in down-regulation of DG-based signalling pathways and perhaps activation of PA-stimulated signalling events. We report here that the simple protozoan amoeba Dictyostelium discoideum appears to contain a single gene encoding a DGK enzyme. This gene, dgkA, encodes a deduced protein that contains three C1-type cysteine-rich repeats, a DGK catalytic domain most closely related to the θ subtype of mammalian DGKs and a C-terminal segment containing a proline/glutamine-rich region and a large aspargine-repeat region. This gene corresponds to a previously reported myosin II heavy chain kinase designated myosin heavy chain-protein kinase C (MHC-PKC), but our analysis clearly demonstrates that this protein does not, as suggested by earlier data, contain a protein kinase catalytic domain. A FLAG-tagged version of DgkA expressed in Dictyostelium displayed robust DGK activity. Earlier studies indicating that disruption of this locus alters myosin II assembly levels in Dictyostelium raise the intriguing possibility that DG and/or PA metabolism may play a role in controlling myosin II assembly in this system.

scholarly journals Functional Alterations in Ciliogenesis-Associated Kinase 1 (CILK1) that Result from Mutations Linked to Juvenile Myoclonic Epilepsy

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 694 ◽  
Author(s):  
Eric J. Wang ◽  
Casey D. Gailey ◽  
David L. Brautigan ◽  
Zheng Fu
Keyword(s):  
Primary Cilium ◽  
Primary Cilia ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Intracellular Localization ◽  
Juvenile Myoclonic Epilepsy ◽  
Myoclonic Epilepsy ◽  
Intestinal Cell ◽  
Pathogenic Mutations ◽  
Cilia Formation

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.

scholarly journals The prevalent I686T human variant and loss of function mutations in the cardiomyocyte-specific kinase gene TNNI3K cause adverse contractility and concentric remodeling in mice

2020 ◽  
Author(s):  
Peiheng Gan ◽  
Catalin Baicu ◽  
Hirofumi Watanabe ◽  
Kristy Wang ◽  
Ge Tao ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Substantial Reduction ◽  
Mutant Mice ◽  
Wall Thickening ◽  
Contractile Reserve ◽  
Mouse Heart ◽  
Loss Of Function ◽  
Kinase Gene ◽  
Adult Heart ◽  
Heart Pathology

Abstract TNNI3K expression worsens disease progression in several mouse heart pathology models. TNNI3K expression also reduces the number of diploid cardiomyocytes, which may be detrimental to adult heart regeneration. However, the gene is evolutionarily conserved, suggesting a beneficial function that has remained obscure. Here, we show that C57BL/6 J-inbred Tnni3k mutant mice develop concentric remodeling, characterized by ventricular wall thickening and substantial reduction of cardiomyocyte aspect ratio. This pathology occurs in mice carrying a Tnni3k null allele, a K489R point mutation rendering the protein kinase-dead, or an allele corresponding to human I686T, the most common human nonsynonymous TNNI3K variant, which is hypomorphic for kinase activity. Mutant mice develop these conditions in the absence of fibrosis or hypertension, implying a primary cardiomyocyte etiology. In culture, mutant cardiomyocytes were impaired in contractility and calcium dynamics and in protein kinase A signaling in response to isoproterenol, indicating diminished contractile reserve. These results demonstrate a beneficial function of TNNI3K in the adult heart that might explain its evolutionary conservation and imply that human TNNI3K variants, in particular the widespread I686T allele, may convey elevated risk for altered heart geometry and hypertrophy.

scholarly journals The Carboxyl-terminal Domain of Atypical Protein Kinase Cζ Binds to Ceramide and Regulates Junction Formation in Epithelial Cells

2009 ◽  
Vol 284 (21) ◽  
pp. 14469-14475 ◽  
Author(s):  
Guanghu Wang ◽  
Kannan Krishnamurthy ◽  
Nagavedi S. Umapathy ◽  
Alexander D. Verin ◽  
Erhard Bieberich
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Atypical Protein Kinase ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Junction Formation ◽  
Carboxyl Terminal

scholarly journals Hypermorphic SERK1 mutations function via a SOBIR1 pathway to activate floral abscission signaling

2018 ◽  
Author(s):  
Isaiah Taylor ◽  
John Baer ◽  
Ryan Calcutt ◽  
John C. Walker
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Negative Regulator ◽  
Peptide Ligand ◽  
Protein Kinase Activity ◽  
Loss Of Function ◽  
Allelic Series ◽  
Kinase Gene ◽  
Floral Organs ◽  
Suppression Effect

AbstractIn Arabidopsis, the abscission of floral organs is regulated by two related receptor-like protein kinases (RLKs), HAESA and HAESA-like 2 (HAE/HSL2). HAE/HSL2, in complex with members of the SERK family of coreceptor protein kinases, are activated by the binding of the proteolytically processed peptide ligand IDA. This leads to expression of genes encoding secreted cell wall remodeling and hydrolase enzymes. hae hsl2 mutants fail to induce expression of these genes and retain floral organs indefinitely. In this paper we report identification of an allelic series of hae hsl2 suppressor mutations in the SERK1 coreceptor protein kinase gene. Genetic and transcriptomic evidence indicates these alleles represent a novel class of gain of function mutations that activate signaling independent of HAE/HSL2. We show that the suppression effect surprisingly does not rely on protein kinase activity of SERK1, and that activation of signaling relies on the RLK gene SOBIR1. The effect of these mutations can be mimicked by loss of function of BIR1, a known negative regulator of SERK-SOBIR1 signaling. These results suggest BIR1 functions to negatively regulate SERK-SOBIR1 signaling during abscission, and that the identified SERK1 mutations likely interfere with this negative regulation.

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