Kinase Non-catalytic C-lobe Domain Research Articles

KNDC1 Is a Predictive Marker of Malignant Transformation in Borderline Ovarian Tumors.

BackgroundFew screening markers for malignant transformation in borderline ovarian tumors (BOT) have been clearly established. The kinase noncatalytic C-lobe domain containing 1 (KNDC1), a brain-specific Ras guanine nucleotide exchange factor, negatively regulates dendrite growth. However, the biological role and underlying mechanism of KNDC1 in human cancers, including ovarian cancer (OC), remain unknown.MethodsGene chip screening was used to detect the expression of KNDC1 mRNA in normal ovarian tissues, BOT tissues, and OC tissues. And results were further validated by RT-qPCR, Western blotting and immunohistochemistry. KNDC1 overexpression and knockdown ovarian cancer cells were established to study the possible pathways that KNDC1 was involved. The effects of KNDC1 on the malignant behaviors of ovarian tumors were also investigated both in vitro and in vivo.ResultsWe observed that the expression of KNDC1 mRNA and KNDC1 protein in OC was significantly downregulated compared with BOT. Subsequent investigation revealed that knockdown of KNDC1 enhanced the proliferation of ovarian cancer cells in vitro via induction of ERK1/2 phosphorylation, whereas reinforcing the expression of KNDC1 attenuated the ERK1/2 activity. Similarly, knockdown of KNDC1 also promoted cell proliferation in vivo. Survival analysis showed that lower KNDC1 predicted a poor progression-free survival (PFS) for patients.ConclusionCollectively, we conclude that KNDC1 might function as a tumor suppressor in ovarian tumors, inhibiting the proliferation of ovarian cells by suppressing ERK1/2 activity and hindering the malignant transformation of BOT.

A whole genome sequence association study of muscle fiber traits in a White Duroc×Erhualian F2 resource population.

ObjectiveMuscle fiber types, numbers and area are crucial aspects associated with meat production and quality. However, there are few studies of pig muscle fibre traits in terms of the detection power, false discovery rate and confidence interval precision of whole-genome quantitative trait loci (QTL). We had previously performed genome scanning for muscle fibre traits using 183 microsatellites and detected 8 significant QTLs in a White Duroc× Erhualian F2 population. The confidence intervals of these QTLs ranged between 11 and 127 centimorgan (cM), which contained hundreds of genes and hampered the identification of QTLs. A whole-genome sequence imputation of the population was used for fine mapping in this study.MethodsA whole-genome sequences association study was performed in the F2 population. Genotyping was performed for 1,020 individuals (19 F0, 68 F1, and 933 F2). The whole-genome variants were imputed and 21,624,800 single nucleotide polymorphisms (SNPs) were identified and examined for associations to 11 longissimus dorsi muscle fiber traits.ResultsA total of 3,201 significant SNPs comprising 7 novel QTLs showing associations with the relative area of fiber type I (I_RA), the fiber number per square centimeter (FN) and the total fiber number (TFN). Moreover, one QTL on pig chromosome 14 was found to affect both FN and TFN. Furthermore, four plausible candidate genes associated with FN (kinase non-catalytic C-lobe domain containing [KNDC1]), TFN (KNDC1), and I_RA (solute carrier family 36 member 4, contactin associated protein like 5, and glutamate metabotropic receptor 8) were identified.ConclusionAn efficient and powerful imputation-based association approach was utilized to identify genes potentially associated with muscle fiber traits. These identified genes and SNPs could be explored to improve meat production and quality via marker-assisted selection in pigs.

Effect of KNDC1 overexpression on the senescence of human umbilical vein endothelial cells.

Kinase non-catalytic C-lobe domain containing 1 (KNDC1) exists in dendrites, guanine nucleotide exchange factor complexes and neuronal cell bodies as a putative protein-protein interaction module that regulates a number of signaling pathways. Previous studies have demonstrated that the knockdown of KNDC1 delays human umbilical vein endothelial cell (HUVEC) senescence. However, the effect of KNDC1 overexpression on HUVEC function remains unclear. In the present study, an adenovirus vector carrying KNDC1 was constructed and then transfected into endothelial cells to observe cell senescence. Furthermore, the effect of KNDC1 overexpression on HUVEC senescence was investigated in vitro and the underlying molecular mechanism was investigated. Senescence-associated β-galactosidase staining was used to determine cellular senescence and reactive oxygen species (ROS) were monitored to detect the level of cell oxidative stress. The mRNA transcription level and protein expression were analyzed by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The results demonstrated that KNDC1 overexpression possibly inhibited HUVEC activity and function and promoted HUVEC senescence. Mechanistic studies demonstrated that KNDC1 triggered a p53-ROS positive feedback loop, which serves a crucial role in regulating senescence. In conclusion, to the best of the authors' knowledge, this is the first time that KNDC1-adenovirus vector inhibition of HUVEC proliferation by activating the p53 signaling pathway has been reported. Theoretically, the results of the present study also support KNDC1 as a therapeutic target for future anti-senescence.

The brain-specific RasGEF very-KIND is required for normal dendritic growth in cerebellar granule cells and proper motor coordination.

Very-KIND/Kndc1/KIAA1768 (v-KIND) is a brain-specific Ras guanine nucleotide exchange factor carrying two sets of the kinase non-catalytic C-lobe domain (KIND), and is predominantly expressed in cerebellar granule cells. Here, we report the impact of v-KIND deficiency on dendritic and synaptic growth in cerebellar granule cells in v-KIND knockout (KO) mice. Furthermore, we evaluate motor function in these animals. The gross anatomy of the cerebellum, including the cerebellar lobules, layered cerebellar cortex and densely-packed granule cell layer, in KO mice appeared normal, and was similar to wild-type (WT) mice. However, KO mice displayed an overgrowth of cerebellar granule cell dendrites, compared with WT mice, resulting in an increased number of dendrites, dendritic branches and terminals. Immunoreactivity for vGluT2 (a marker for excitatory presynapses of mossy fiber terminals) was increased in the cerebellar glomeruli of KO mice, compared with WT mice. The postsynaptic density around the terminals of mossy fibers was also increased in KO mice. Although there were no significant differences in locomotor ability between KO and WT animals in their home cages or in the open field, young adult KO mice had an increased grip strength and a tendency to exhibit better motor performance in balance-related tests compared with WT animals. Taken together, our results suggest that v-KIND is required for compact dendritic growth and proper excitatory synaptic connections in cerebellar granule cells, which are necessary for normal motor coordination and balance.

Membrane Targeting of the Spir·Formin Actin Nucleator Complex Requires a Sequential Handshake of Polar Interactions

Spir and formin (FMN)-type actin nucleators initiate actin polymerization at vesicular membranes necessary for long range vesicular transport processes. Here we studied in detail the membrane binding properties and protein/protein interactions that govern the assembly of the membrane-associated Spir·FMN complex. Using biomimetic membrane models we show that binding of the C-terminal Spir-2 FYVE-type zinc finger involves both the presence of negatively charged lipids and hydrophobic contributions from the turret loop that intrudes the lipid bilayer. In solution, we uncovered a yet unknown intramolecular interaction between the Spir-2 FYVE-type domain and the N-terminal kinase non-catalytic C-lobe domain (KIND) that could not be detected in the membrane-bound state. Interestingly, we found that the intramolecular Spir-2 FYVE/KIND and the trans-regulatory Fmn-2-FSI/Spir-2-KIND interactions are competitive. We therefore characterized co-expressed Spir-2 and Fmn-2 fluorescent protein fusions in living cells by fluorescence cross-correlation spectroscopy. The data corroborate a model according to which Spir-2 exists in two different states, a cytosolic monomeric conformation and a membrane-bound state in which the KIND domain is released and accessible for subsequent Fmn-2 recruitment. This sequence of interactions mechanistically couples membrane binding of Spir to the recruitment of FMN, a pivotal step for initiating actin nucleation at vesicular membranes.

KNDC1 knockdown protects human umbilical vein endothelial cells from senescence.

KNDC1 (kinase noncatalytic C-lobe domain containing 1), a brain-specific Ras guanine nucleotide exchange factor, controls the negative regulation of neuronal dendrite growth. However, the effect of KNDC1 on cellular senescence remains to be elucidated. The present study investigated the impact of KNDC1 knockdown on human endothelial cell senescence and the mechanisms underlying this effect. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as a model of biological aging. Senescence-associated β-galactosidase staining was used to detect cellular senescence and flow cytometry was employed to determine cell cycle progression. Quantitative polymerase chain reaction (qPCR) and western blot analysis were utilized to investigate mRNA transcription and protein expression. In the HUVECs, a senescence-like phenotypes developed with increasing passage number in vitro, which were associated with a progressive increase in the transcription and expression of KNDC1. KNDC1 knockdown promoted cell proliferation and partially reversed cellular senescence and cell cycle arrest in the G0/G1 phase in aging HUVECs. Investigations into the mechanism underlying this effect demonstrated that KNDC1 knockdown promoted HUVEC proliferation via the extracellular signal-regulated kinase signaling pathway and delayed HUVEC senescence by inhibiting the p53-p21-p16 transduction cascade. In addition, the promotion of the capillary tube network formation and the increased expression of endothelial nitric oxide synthase revealed that the activity and function of endothelial cells were enhanced. In conclusion, KNDC1 knockdown delayed endothelial cell senescence and promoted HUVEC activity and function. These results demonstrated that KNDC1 may be a novel therapeutic target for the development of agents to extend human life.

Molecular architecture of the Spire–actin nucleus and its implication for actin filament assembly

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire-actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, we find that preformed, isolated Spire-actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs--even a single WH2 repeat--sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem.

Molecular Basis of Actin Nucleation Factor Cooperativity: CRYSTAL STRUCTURE OF THE SPIR-1 KINASE NON-CATALYTIC C-LOBE DOMAIN (KIND)·FORMIN-2 FORMIN SPIR INTERACTION MOTIF (FSI) COMPLEX

The distinct actin nucleation factors of the Spir and formin subgroup families cooperate in actin nucleation. The Spir/formin cooperativity has been identified to direct two essential steps in mammalian oocyte maturation, the asymmetric spindle positioning and polar body extrusion during meiosis. Understanding the nature and regulation of the Spir/Fmn cooperation is an important requirement to comprehend mammalian reproduction. Recently we dissected the structural elements of the Spir and Fmn family proteins, which physically link the two actin nucleation factors. The trans-regulatory interaction is mediated by the Spir kinase non-catalytic C-lobe domain (KIND) and the C-terminal formin Spir interaction motif (FSI). The interaction inhibits formin nucleation activity and enhances the Spir activity. To get insights into the molecular mechanism of the Spir/Fmn interaction, we determined the crystal structure of the KIND domain alone and in complex with the C-terminal Fmn-2 FSI peptide. Together they confirm the proposed structural homology of the KIND domain to the protein kinase fold and reveal the basis of the Spir/formin interaction. The complex structure showed a large interface with conserved and positively charged residues of the Fmn FSI peptide mediating major contacts to an acidic groove on the surface of KIND. Protein interaction studies verified the electrostatic nature of the interaction. The data presented here provide the molecular basis of the Spir/formin interaction and give a first structural view into the mechanisms of actin nucleation factor cooperativity.

Structure and function of the interacting domains of Spire and Fmn-family formins

Evidence for cooperation between actin nucleators is growing. The WH2-containing nucleator Spire and the formin Cappuccino interact directly, and both are essential for assembly of an actin mesh during Drosophila oogenesis. Their interaction requires the kinase noncatalytic C-lobe domain (KIND) domain of Spire and the C-terminal tail of the formin. Here we describe the crystal structure of the KIND domain of human Spir1 alone and in complex with the tail of Fmn2, a mammalian ortholog of Cappuccino. The KIND domain is structurally similar to the C-lobe of protein kinases. The Fmn2 tail is coordinated in an acidic cleft at the base of the domain that appears to have evolved via deletion of a helix from the canonical kinase fold. Our functional analysis of Cappuccino reveals an unexpected requirement for its tail in actin assembly. In addition, we find that the KIND/tail interaction blocks nucleation by Cappuccino and promotes its displacement from filament barbed ends providing insight into possible modes of cooperation between Spire and Cappuccino.

Interaction between very‐KIND Ras guanine exchange factor and microtubule‐associated protein 2, and its role in dendrite growth – structure and function of the second kinase noncatalytic C‐lobe domain

The kinase noncatalytic C-lobe domain (KIND) is a putative protein-protein interaction module. Four KIND-containing proteins, Spir-2 (actin-nuclear factor), PTPN13 (protein tyrosine phosphatase), FRMPD2 (scaffold protein) and very-KIND (v-KIND) (brain-specific Ras guanine nucleotide exchange factor), have been identified to date. Uniquely, v-KIND has two KINDs (i.e. KIND1 and KIND2), whereas the other three proteins have only one. The functional role of KIND, however, remains unclear. We previously demonstrated that v-KIND interacts with the high-molecular weight microtubule-associated protein 2 (MAP2), a dendritic microtubule-associated protein, leading to negative regulation of neuronal dendrite growth. In the present study, we analyzed the structure-function relationships of the v-KIND-MAP2 interaction by generating a series of mutant constructs. The interaction with endogenous MAP2 in mouse cerebellar granule cells was specific to v-KIND KIND2, but not KIND1, and was not observed for the KINDs from other KIND-containing proteins. The binding core modules critical for the v-KIND-MAP2 interaction were defined within 32 residues of the mouse v-KIND KIND2 and 43 residues of the mouse MAP2 central domain. Three Leu residues at amino acid positions 461, 474 and 477 in the MAP2-binding core module of KIND2 contributed to the interaction. The MAP2-binding core module itself promoted dendrite branching as a dominant-negative regulator of v-KIND in hippocampal neurons. The results reported in the present study demonstrate the structural and functional determinant underlying the v-KIND-MAP2 interaction that controls dendrite arborization patterns.

Cadherin-like 24 (CDH24); solute carrier family 15 (oligopeptide transporter), member 1 (SLC15A); kinase non-catalytic C-lobe domain (KIND) containing 1 (KNDC1); protein tyrosine phosphatase, receptor type, T (PTPRT); glutamate receptor, ionotropic, N-methyl D-aspartate-like 1B (GRINL1B); G protein–coupled receptor 123 (GPR123); G protein–coupled receptor 183 (GPR183; EBI2); protocadherin 24 (PCDH24; PC-LKC); fms-related tyrosine kinase 3 (FLT3); nucleophosmin (nucleolar phosphoprotein B23, numatrin) (NPM1)

Cadherin-like 24 (CDH24); solute carrier family 15 (oligopeptide transporter), member 1 (SLC15A); kinase non-catalytic C-lobe domain (KIND) containing 1 (KNDC1); protein tyrosine phosphatase, receptor type, T (PTPRT); glutamate receptor, ionotropic, N-methyl D-aspartate-like 1B (GRINL1B); G protein–coupled receptor 123 (GPR123); G protein–coupled receptor 183 (GPR183; EBI2); protocadherin 24 (PCDH24; PC-LKC); fms-related tyrosine kinase 3 (FLT3); nucleophosmin (nucleolar phosphoprotein B23, numatrin) (NPM1)

Regulatory interactions between two actin nucleators, Spire and Cappuccino

Spire and Cappuccino are actin nucleation factors that are required to establish the polarity of Drosophila melanogaster oocytes. Their mutant phenotypes are nearly identical, and the proteins interact biochemically. We find that the interaction between Spire and Cappuccino family proteins is conserved across metazoan phyla and is mediated by binding of the formin homology 2 (FH2) domain from Cappuccino (or its mammalian homologue formin-2) to the kinase noncatalytic C-lobe domain (KIND) from Spire. In vitro, the KIND domain is a monomeric folded domain. Two KIND monomers bind each FH2 dimer with nanomolar affinity and strongly inhibit actin nucleation by the FH2 domain. In contrast, formation of the Spire–Cappuccino complex enhances actin nucleation by Spire. In Drosophila oocytes, Spire localizes to the cortex early in oogenesis and disappears around stage 10b, coincident with the onset of cytoplasmic streaming.

PEg6, a Spire family member, is a maternal gene encoding a vegetally localized mRNA in Xenopus embryos

In Xenopus, during oocyte maturation and the segmentation period, cell cycle progression is independent of new transcription, but requires de novo translation. This suggests that the completion of oocyte maturation and then the rapid cell division period is controlled exclusively at a post-transcriptional level by specific gene products. To isolate these maternal genes, a differential screening of a Xenopus egg cDNA library was performed. Several cDNAs were isolated which correspond to mRNA polyadenylated in eggs and deadenylated in embryos, and these constitute the founders members of the Eg family of mRNAs. We report here the characterization of Eg6 mRNA as a novel maternal gene expressed in Xenopus egg until gastrula stage. The Eg6 transcript is initially concentrated in the vegetal cytoplasm of the egg, and later the distribution of the transcript marks the posterior vegetal end of developing embryos. pEg6 is a multidomain protein with a kinase non-catalytic C-lobe domain of unknown function, a cluster of four WH2 (Wiskott-Aldrich syndrome protein homology 2) domains and a modified FYVE zinc-finger motif. The amino acid sequence of pEg6 is related to PEM-5 (posterior end mark-5), from an ascidian maternal mRNA, and spire, a Drosophila protein required to establish dorsal-ventral and anterior-posterior axes of polarity and recently described as an actin nucleation factor. In Xenopus and Schizosaccharomyces pombe cells pEg6 expression induces filamentous actin clusters and is associated with vesicular structure. These data suggest that pEg6 acts as a vegetally localized factor contributing to the actin nucleation process during Xenopus early development.

Very-KIND is a novel nervous system specific guanine nucleotide exchange factor for Ras GTPases

The kinase non-catalytic c-lobe domain (KIND) evolved from the catalytic protein kinase fold into a potential protein interaction module for signalling proteins. Spir family actin organizers and the non-receptor phosphatase type 13 (PTP type 13) encode a KIND domain in the very N-terminal parts of the proteins. Here we report the characterization and cloning of a third member of the KIND protein family, which we have named very-KIND (VKIND) because of its two KIND domains. Like the other members of the protein family, VKIND has a KIND domain at the N-terminus. A second KIND domain is located in the central part of the protein. The C-terminal half encodes a guanine nucleotide exchange factor motif for Ras-like GTPases (RasGEF) and a RasGEF N-terminal module (RasGEFN). There is only one VKIND gene in the mammalian genomes and up to now we have found the gene only in vertebrates. During mouse embryogenesis the VKIND gene was specifically expressed in the developing nervous system. In adult mice Northern hybridizations revealed high expression only in brain. Low expression could be detected in ovary. In situ hybridizations showed a specific expression of VKIND in neuronal cells of the granular and Purkinje cell layers of the cerebellum.

The KIND module: a putative signalling domain evolved from the C lobe of the protein kinase fold

A novel putative interaction domain – KIND (kinase non-catalytic C-lobe domain) – has been identified as being similar to the C-terminal protein kinase catalytic fold (C lobe). Its presence at the N terminus of signalling proteins and the absence of the active-site residues in the catalytic and activation loops suggest that it folds independently and is likely to be non-catalytic. The occurrence of the novel domain only in metazoa implies that it has evolved from the catalytic protein kinase domain into an interaction domain possibly by keeping the substrate-binding features.