scholarly journals Protein Kinase MARK/PAR-1 Is Required for Neurite Outgrowth and Establishment of Neuronal Polarity

2002 ◽  
Vol 13 (11) ◽  
pp. 4013-4028 ◽  
Author(s):  
Jacek Biernat ◽  
Yong-Zhong Wu ◽  
Thomas Timm ◽  
Qingyi Zheng-Fischhöfer ◽  
Eckhard Mandelkow ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Tau Protein ◽  
Point Mutations ◽  
Dominant Negative ◽  
Neuronal Polarity ◽  
Dominant Negative Mutant ◽  
Cell Models ◽  
Alzheimer Brain ◽  
Repeat Domain ◽  
Cell Processes

Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate certain sites in tau protein (KXGS motifs in the repeat domain). This type of phosphorylation is enhanced in abnormal tau from Alzheimer brain tissue and causes the detachment of tau from microtubules. MARK-related kinases (PAR-1 and KIN1) occur in various organisms and are involved in establishing and maintaining cell polarity. Herein, we report the ability of MARK2 to affect the differentiation and outgrowth of cell processes from neuroblastoma and other cell models. MARK2 phosphorylates tau protein at the KXGS motifs; this results in the detachment of tau from microtubules and their destabilization. The formation of neurites in N2a cells is blocked if MARK2 is inactivated, either by transfecting a dominant negative mutant, or by MARK2 inhibitors such as hymenialdisine. Alternatively, neurites are blocked if the target KXGS motifs on tau are rendered nonphosphorylatable by point mutations. The results suggest that MARK2 contributes to the plasticity of microtubules needed for neuronal polarity and the growth of neurites.

scholarly journals Atypical Protein Kinase C Is Involved in the Evolutionarily Conserved Par Protein Complex and Plays a Critical Role in Establishing Epithelia-Specific Junctional Structures

2001 ◽  
Vol 152 (6) ◽  
pp. 1183-1196 ◽  
Author(s):  
Atsushi Suzuki ◽  
Tomoyuki Yamanaka ◽  
Tomonori Hirose ◽  
Naoyuki Manabe ◽  
Keiko Mizuno ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Polarity ◽  
Protein Complex ◽  
Mdck Cells ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Surface Polarity ◽  
C Elegans ◽  
Evolutionarily Conserved

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

scholarly journals Nuclear Import of IκBα Is Accomplished by a Ran-Independent Transport Pathway

2000 ◽  
Vol 20 (5) ◽  
pp. 1571-1582 ◽  
Author(s):  
Shrikesh Sachdev ◽  
Sriparna Bagchi ◽  
Donna D. Zhang ◽  
Angela C. Mings ◽  
Mark Hannink
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Nuclear Pore ◽  
Transport Pathway ◽  
Repeat Domain ◽  
Nuclear Export Receptor

ABSTRACT The inhibitor of kappa B alpha (IκBα) protein is able to shuttle between the cytoplasm and the nucleus. We have utilized a combination of in vivo and in vitro approaches to provide mechanistic insight into nucleocytoplasmic shuttling by IκBα. IκBα contains multiple functional domains that contribute to shuttling of IκBα between the cytoplasm and the nucleus. Nuclear import of IκBα is mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of IκBα is temperature and ATP dependent and is blocked by a dominant-negative mutant of importin β. However, in contrast to classical nuclear import pathways, nuclear import of IκBα is independent of soluble cytosolic factors and is not blocked by the dominant-negative RanQ69L protein. Nuclear export of IκBα is mediated by an N-terminal nuclear export sequence. Nuclear export of IκBα requires the CRM1 nuclear export receptor and is blocked by the dominant-negative RanQ69L protein. Our results are consistent with a model in which nuclear import of IκBα is mediated through direct interactions with components of the nuclear pore complex, while nuclear export of IκBα is mediated via a CRM1-dependent pathway.

scholarly journals Siah-1 N-Terminal RING Domain Is Required for Proteolysis Function, and C-Terminal Sequences Regulate Oligomerization and Binding to Target Proteins

1999 ◽  
Vol 19 (1) ◽  
pp. 724-732 ◽  
Author(s):  
Gang Hu ◽  
Eric R. Fearon
Keyword(s):  
Cell Fate ◽  
Point Mutations ◽  
Dominant Negative ◽  
Ring Domain ◽  
Dominant Negative Mutant ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Target Proteins ◽  
Proteasome Pathway ◽  
Seven In Absentia

ABSTRACT The Drosophila seven in absentia (sina) gene was initially discovered because its inactivation leads to R7 photoreceptor defects. Recent data indicate that Sina binds to the Sevenless pathway protein Phyllopod, and together they mediate degradation of Tramtrack, a transcriptional repressor of R7 cell fate. Independent studies have shown that Sina and its highly related mammalian homologues Siah-1 and Siah-2 bind to the DCC (deleted in colorectal cancer) protein and promote its proteolysis via the ubiquitin-proteasome pathway. To determine the roles of mammalian Siahs in proteolysis and their interactions with target proteins, we sought to define Siah-1 domains critical for regulation of DCC. Mutant Siah-1 proteins, harboring missense mutations in the carboxy (C)-terminal domain analogous to those present in Drosophila sinaloss-of-function alleles, failed to promote DCC degradation. Point mutations and deletion of the amino (N)-terminal RING finger domain of Siah-1 abrogated its ability to promote DCC proteolysis. In the course of defining Siah-1 sequences required for DCC degradation, we found that Siah-1 is itself rapidly degraded via the proteasome pathway, and RING domain mutations stabilized the Siah-1 protein. Siah-1 was found to oligomerize with itself and other Sina and Siah proteins via C-terminal sequences. Finally, evidence that endogenous Siah-1 regulates DCC proteolysis in cells was obtained through studies of an apparent dominant negative mutant of Siah-1, as well as via an antisense approach. The data indicate that the Siah-1 N-terminal RING domain is required for its proteolysis function, while the C-terminal sequences regulate oligomerization and binding to target proteins, such as DCC.

scholarly journals Evolution of gonadotropin deficiency in a patient with type II autosomal dominant GH deficiency

2006 ◽  
Vol 155 (6) ◽  
pp. 793-799 ◽  
Author(s):  
James P G Turton ◽  
Charles R Buchanan ◽  
Iain C A F Robinson ◽  
Simon J B Aylwin ◽  
Mehul T Dattani
Keyword(s):  
Gh Deficiency ◽  
Semen Quality ◽  
Point Mutations ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Type Ii ◽  
Clinical Observations ◽  
Prolactin Response ◽  
Lost To Follow Up

Background: Type II isolated GH deficiency (IGHD type II) is caused by dominant negative splicing or point mutations of the GH-1 gene. Studies have suggested that dominant mutant GH forms prevent the secretion of wild-type GH, resulting in eventual cell death; surprisingly, some patients with these GH mutations develop other hormonal deficiencies (ACTH, TSH). Subjects: The proband presented at the age of 2.3 years with IGHD. His father, also known to have been treated for IGHD as a child, had subsequently been lost to follow-up, having remained without treatment during this time. At re-evaluation at the age of 38 years, he complained of lack of stamina and poor libido. Clinical and biochemical assessment confirmed severe GHD, borderline ACTH insufficiency, suboptimal basal and stimulated gonadotropins, and a poor prolactin response to provocation. The basal testosterone concentration was low, and he complained of secondary infertility. Magnetic resonance imaging revealed anterior pituitary hypoplasia in both patients. Genetic testing revealed a heterozygous splicing mutation in GH-1 (intervening sequence-3 + 1G>A) in both patients, known to cause IGHD type II. Interventions: The proband showed an excellent growth response to recombinant human GH (rhGH). His father, also treated with rhGH, showed improved quality of life on rhGH, but testosterone concentrations continued to decline, necessitating treatment with testosterone with symptomatic benefit but no improvement in semen quality. Conclusions: This case supports recent experimental and clinical observations suggesting that the cytotoxicity associated with accumulation of dominant negative mutant 17.5 kDa GH causes a form of GHD that can evolve into multiple hormone deficiencies. Hence, patients diagnosed initially with IGHD type II require continued long-term clinical follow-up.

scholarly journals The Development of Cell Processes Induced by tau Protein Requires Phosphorylation of Serine 262 and 356 in the Repeat Domain and Is Inhibited by Phosphorylation in the Proline-rich Domains

1999 ◽  
Vol 10 (3) ◽  
pp. 727-740 ◽  
Author(s):  
Jacek Biernat ◽  
Eva-Maria Mandelkow
Keyword(s):  
Tau Protein ◽  
Approximate Model ◽  
Necessary Condition ◽  
Sf9 Cells ◽  
Microtubule Associated Proteins ◽  
Repeat Domain ◽  
Phosphorylation Pattern ◽  
Associated Proteins ◽  
Cell Processes ◽  
A Minor

The differentiation of neurons and the outgrowth of neurites depends on microtubule-associated proteins such as tau protein. To study this process, we have used the model of Sf9 cells, which allows efficient transfection with microtubule-associated proteins (via baculovirus vectors) and observation of the resulting neurite-like extensions. We compared the phosphorylation of tau23 (the embryonic form of human tau) with mutants in which critical phosphorylation sites were deleted by mutating Ser or Thr residues into Ala. One can broadly distinguish two types of sites, the KXGS motifs in the repeats (which regulate the affinity of tau to microtubules) and the SP or TP motifs in the domains flanking the repeats (which contain epitopes for antibodies diagnostic of Alzheimer’s disease). Here we report that both types of sites can be phosphorylated by endogenous kinases of Sf9 cells, and that the phosphorylation pattern of the transfected tau is very similar to that of neurons, showing that Sf9 cells can be regarded as an approximate model for the neuronal balance between kinases and phosphatases. We show that mutations in the repeat domain and in the flanking domains have opposite effects. Mutations of KXGS motifs in the repeats (Ser262, 324, and 356) strongly inhibit the outgrowth of cell extensions induced by tau, even though this type of phosphorylation accounts for only a minor fraction of the total phosphate. This argues that the temporary detachment of tau from microtubules (by phosphorylation at KXGS motifs) is a necessary condition for establishing cell polarity at a critical point in space or time. Conversely, the phosphorylation at SP or TP motifs represents the majority of phosphate (>80%); mutations in these motifs cause an increase in cell extensions, indicating that this type of phosphorylation retards the differentiation of the cells.

scholarly journals An Assembly-incompetent Mutant Establishes a Requirement for Dynamin Self-assembly in Clathrin-mediated Endocytosis In Vivo

2004 ◽  
Vol 15 (5) ◽  
pp. 2243-2252 ◽  
Author(s):  
Byeong Doo Song ◽  
Defne Yarar ◽  
Sandra L. Schmid
Keyword(s):  
Self Assembly ◽  
Point Mutations ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Gtpase Activity ◽  
Insight Into ◽  
Assay Conditions ◽  
In Cells

Dynamin GTPase activity is required for its biological function in clathrin-mediated endocytosis; however, the role of self-assembly has not been unambiguously established. Indeed, overexpression of a dynamin mutant, Dyn1-K694A, with impaired ability to self-assemble has been shown to stimulate endocytosis in HeLa cells (Sever et al., Nature 1999, 398, 481). To identify new, assembly-incompetent mutants of dynamin 1, we made point mutations in the GTPase effector/assembly domain (GED) and tested for their effects on self-assembly and clathrin-mediated endocytosis. Mutation of three residues, I690, K694, and I697, suggests that interactions with an amphipathic helix in GED are required for self-assembly. In particular, Dyn1-I690K failed to exhibit detectable assembly-stimulated GTPase activity under all assay conditions. Overexpression of this assembly-incompetent mutant inhibited transferrin endocytosis as potently as the GTPase-defective dominant-negative mutant, Dyn1-K44A. However, worm-like endocytic intermediates accumulated in cells expressing Dyn1-I690K that were structurally distinct from long tubules that accumulated in cells expressing Dyn1-K44A. Together these results provide new structural insight into the role of GED in self-assembly and assembly-stimulated GTPase activity and establish that dynamin self-assembly is essential for clathrin-mediated endocytosis.

SOX-18 controls endothelial-specific claudin-5 gene expression and barrier function

2008 ◽  
Vol 294 (2) ◽  
pp. H891-H900 ◽  
Author(s):  
Ruud D. Fontijn ◽  
Oscar L. Volger ◽  
Joost O. Fledderus ◽  
Arie Reijerkerk ◽  
Helga E. de Vries ◽  
...  
Keyword(s):  
Gene Expression ◽  
Barrier Function ◽  
Transcriptional Control ◽  
Expression Patterns ◽  
Point Mutations ◽  
High Mobility ◽  
Dominant Negative ◽  
Endothelial Barrier ◽  
Dominant Negative Mutant ◽  
Dependent Manner

Members of the claudin family constitute tight junction strands and are major determinants in specificity and selectivity of paracellular barriers. Transcriptional control of claudin gene expression is essential to establish individual claudin expression patterns and barrier properties. Using full genome expression profiling, we now identify sex-determining region Y-box (SOX)-18, a member of the SOX family of high-mobility group box transcription factors, as one of the most differentially induced genes during establishment of the endothelial barrier. We show that overexpression of SOX-18 and a dominant-negative mutant thereof, as well as SOX-18 silencing, greatly affect levels of claudin-5 (CLDN5). The relevance of an evolutionary conserved SOX-binding site in the CLDN5 promoter is shown using sequential promoter deletions, as well as point mutations. Furthermore, SOX-18 silencing abrogates endothelial barrier function, as measured by electric cell-substrate impedance sensing. Thus an obligatory role for SOX-18 in the regulation of CLDN5 gene expression in an endothelial-specific and cell density-dependent manner is established, as well as a crucial, nonredundant role for specifically SOX-18 in the formation of the endothelial barrier.

scholarly journals Targeting fibroblast growth factor receptors to combat aggressive ependymoma

2021 ◽  
Author(s):  
Daniela Lötsch ◽  
Dominik Kirchhofer ◽  
Bernhard Englinger ◽  
Li Jiang ◽  
Konstantin Okonechnikov ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Molecular Mechanisms ◽  
Radial Glia ◽  
Growth Factor Receptors ◽  
Dominant Negative ◽  
Mrna Levels ◽  
Fibroblast Growth ◽  
Cell Models ◽  
Fibroblast Growth Factor Receptors

AbstractEpendymomas (EPN) are central nervous system tumors comprising both aggressive and more benign molecular subtypes. However, therapy of the high-risk subtypes posterior fossa group A (PF-A) and supratentorial RELA-fusion positive (ST-RELA) is limited to gross total resection and radiotherapy, as effective systemic treatment concepts are still lacking. We have recently described fibroblast growth factor receptors 1 and 3 (FGFR1/FGFR3) as oncogenic drivers of EPN. However, the underlying molecular mechanisms and their potential as therapeutic targets have not yet been investigated in detail. Making use of transcriptomic data across 467 EPN tissues, we found that FGFR1 and FGFR3 were both widely expressed across all molecular groups. FGFR3 mRNA levels were enriched in ST-RELA showing the highest expression among EPN as well as other brain tumors. We further identified high expression levels of fibroblast growth factor 1 and 2 (FGF1, FGF2) across all EPN subtypes while FGF9 was elevated in ST-EPN. Interrogation of our EPN single-cell RNA-sequencing data revealed that FGFR3 was further enriched in cycling and progenitor-like cell populations. Corroboratively, we found FGFR3 to be predominantly expressed in radial glia cells in both mouse embryonal and human brain datasets. Moreover, we detected alternative splicing of the FGFR1/3-IIIc variant, which is known to enhance ligand affinity and FGFR signaling. Dominant-negative interruption of FGFR1/3 activation in PF-A and ST-RELA cell models demonstrated inhibition of key oncogenic pathways leading to reduced cell growth and stem cell characteristics. To explore the feasibility of therapeutically targeting FGFR, we tested a panel of FGFR inhibitors in 12 patient-derived EPN cell models revealing sensitivity in the low-micromolar to nano-molar range. Finally, we gain the first clinical evidence for the activity of the FGFR inhibitor nintedanib in the treatment of a patient with recurrent ST-RELA. Together, these preclinical and clinical data suggest FGFR inhibition as a novel and feasible approach to combat aggressive EPN.

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