scholarly journals RhoA-Binding Kinase α Translocation Is Facilitated by the Collapse of the Vimentin Intermediate Filament Network

1998 ◽  
Vol 18 (11) ◽  
pp. 6325-6339 ◽  
Author(s):  
Wun-Chey Sin ◽  
Xiang-Qun Chen ◽  
Thomas Leung ◽  
Louis Lim
Keyword(s):  
Structural Integrity ◽  
Morphological Changes ◽  
Dominant Negative ◽  
Cell Periphery ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Complex Process ◽  
Rho Effectors ◽  
Filament Network

ABSTRACT The regulation of morphological changes in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). The putative effector of RhoA, RhoA-binding kinase α (ROKα), is a serine/threonine kinase that has been implicated in the reorganization of actin filaments and in myosin contractility. Here, we show that ROKα also directly affects the structural integrity of IFs. Overexpression of active ROKα, like that of RhoA, caused the collapse of filamentous vimentin, a type III IF. A RhoA-binding-deficient, kinase-inactive ROKα inhibited the collapse of vimentin IFs induced by RhoA in HeLa cells. In vitro, ROKα bound and phosphorylated vimentin at its head-rod domain, thereby inhibiting the assembly of vimentin. ROKα colocalized predominantly with the filamentous vimentin network, which remained intact in serum-starved cells. Treatment of cells with vinblastine, a microtubule-disrupting agent, also resulted in filamentous vimentin collapse and concomitant ROKα translocation to the cell periphery. ROKα translocation did not occur when the vimentin network remained intact in vinblastine-treated cells at 4°C or in the presence of the dominant-negative RhoAN19 mutant. Transient translocation of ROKα was also observed in cells subjected to heat shock, which caused the disassembly of the vimentin network. Thus, the translocation of ROKα to the cell periphery upon overexpression of RhoAV14 or growth factor treatment is associated with disassembly of vimentin IFs. These results indicate that Rho effectors known to act on microfilaments may be involved in regulating the assembly of IFs. Vimentin when phosphorylated also exhibits reduced affinity for the inactive ROKα. The translocation of ROKα from IFs to the cell periphery upon action by activated RhoA and ROKα suggests that ROKα may initiate its own cascade of activation.

Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

2008 ◽  
Vol 415 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Meghna Thakur ◽  
Pradip K. Chakraborti
Keyword(s):  
Protein Kinases ◽  
Solid Phase ◽  
Morphological Changes ◽  
Binding Assays ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Vitro Binding ◽  
Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

scholarly journals Insulin-Induced Phosphorylation and Activation of Cyclic Nucleotide Phosphodiesterase 3B by the Serine-Threonine Kinase Akt

1999 ◽  
Vol 19 (9) ◽  
pp. 6286-6296 ◽  
Author(s):  
Tadahiro Kitamura ◽  
Yukari Kitamura ◽  
Shoji Kuroda ◽  
Yasuhisa Hino ◽  
Miwa Ando ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Second Messengers ◽  
Dominant Negative ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Serine Threonine Kinase ◽  
Intact Cells ◽  
Threonine Kinase

ABSTRACT Cyclic nucleotide phosphodiesterase (PDE) is an important regulator of the cellular concentrations of the second messengers cyclic AMP (cAMP) and cGMP. Insulin activates the 3B isoform of PDE in adipocytes in a phosphoinositide 3-kinase-dependent manner; however, downstream effectors that mediate signaling to PDE3B remain unknown. Insulin-induced phosphorylation and activation of endogenous or recombinant PDE3B in 3T3-L1 adipocytes have now been shown to be inhibited by a dominant-negative mutant of the serine-threonine kinase Akt, suggesting that Akt is necessary for insulin-induced phosphorylation and activation of PDE3B. Serine-273 of mouse PDE3B is located within a motif (RXRXXS) that is preferentially phosphorylated by Akt. A mutant PDE3B in which serine-273 was replaced by alanine was not phosphorylated either in response to insulin in intact cells or by purified Akt in vitro. In contrast, PDE3B mutants in which alanine was substituted for either serine-296 or serine-421, each of which lies within a sequence (RRXS) preferentially phosphorylated by cAMP-dependent protein kinase, were phosphorylated by Akt in vitro or in response to insulin in intact cells. Moreover, the serine-273 mutant of PDE3B was not activated by insulin when expressed in adipocytes. These results suggest that PDE3B is a physiological substrate of Akt and that Akt-mediated phosphorylation of PDE3B on serine-273 is important for insulin-induced activation of PDE3B.

scholarly journals Regulation of the tumour suppressor Fbw7α by PKC-dependent phosphorylation and cancer-associated mutations

2010 ◽  
Vol 432 (1) ◽  
pp. 77-87 ◽  
Author(s):  
Joanne Durgan ◽  
Peter J. Parker
Keyword(s):  
Mammalian Cells ◽  
Tumour Suppressor ◽  
Dependent Manner ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Pkc Protein Kinase C ◽  
Specific Regulation ◽  
The Relationship ◽  
Substrate Binding Domain

Fbw7 (F-box WD40 protein 7) is a major tumour suppressor, which mediates the degradation of several potent oncogenes. PKC (protein kinase C) comprises a serine/threonine kinase family that can promote transformation when dysregulated. In the present study, we investigated the relationship between Fbw7 and PKC. Multiple members of the PKC superfamily interact with the substrate-binding domain of Fbw7. However, we find no evidence for Fbw7-mediated degradation of PKC. Instead, we demonstrate that Fbw7 is a novel substrate for PKC. Two residues within the isoform-specific N-terminus of Fbw7α are phosphorylated in a PKC-dependent manner, both in vitro and in mammalian cells (Ser10 and Ser18). Mutational analyses reveal that phosphorylation of Fbw7α at Ser10 can regulate its nuclear localization. Cancer-associated mutations in nearby residues (K11R and the addition of a proline residue at position 16) influence Fbw7α localization in a comparable manner, suggesting that mislocalization of this protein may be of pathological significance. Together these results provide evidence for both physical and functional interactions between the PKC and Fbw7 families, and yield insights into the isoform-specific regulation of Fbw7α.

scholarly journals WNK4 Kinase: from structure to physiology

2021 ◽  
Author(s):  
Adrian Rafael Murillo-de-Ozores ◽  
Alejandro Rodriguez-Gama ◽  
Hector Carbajal-Contreras ◽  
Gerardo Gamba ◽  
Maria Castaneda-Bueno
Keyword(s):  
Oxidative Stress ◽  
Mouse Models ◽  
Serine Threonine Kinase ◽  
Gene Encoding ◽  
Threonine Kinase ◽  
Physiological Conditions ◽  
Different Levels ◽  
Familial Hyperkalemic Hypertension

With No Lysine (K) kinase 4 (WNK4) belongs to a serine-threonine kinase family characterized by the atypical positioning of its catalytic lysine. Despite the fact that WNK4 has been found in many tissues, the majority of its study has revolved around its function in the kidney, specifically as a positive regulator of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the nephron. This is explained by the description of gain-of-function mutations in the gene encoding WNK4 that cause Familial Hyperkalemic Hypertension (FHHt). This disease is mainly driven by increased downstream activation of the Ste20-related Proline Alanine Rich Kinase (SPAK)/Oxidative Stress Responsive Kinase 1 (OSR1)-NCC pathway, which increases salt reabsorption in the DCT and indirectly impairs renal K+ secretion. Here, we review the large volume of information that has accumulated about different aspects of WNK4 function. We first review the knowledge on WNK4 structure and enumerate the functional domains and motifs that have been characterized. Then, we discuss WNK4 physiological functions based on the information obtained from in vitro studies and from a diverse set of genetically modified mouse models with altered WNK4 function. We then review in vitro and in vivo evidence on the different levels of regulation of WNK4. Finally, we go through the evidence that has suggested how different physiological conditions act through WNK4 to modulate NCC activity.

Arabidopsis MLK3, a Plant-specific Casein Kinase 1, Negatively Regulated Flowering and Phosphorylated Histone H3 in vivo

2019 ◽  
Author(s):  
Zhen Wang ◽  
Junmei Kang ◽  
Shangang Jia ◽  
Tiejun Zhang ◽  
Zhihai Wu ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Histone H3 ◽  
Casein Kinase ◽  
Kinase Assay ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
Altered Expression ◽  
Casein Kinase 1 ◽  
Threonine Kinase

Abstract Background: Casein kinase 1 (CK1) family members are highly conserved serine/threonine kinase present in most eukaryotes with multiple biological functions. Arabidopsis MUT9-like kinases ( MLKs ) belong to a clade CK1 specific to the plant kingdom and have been implicated collectively in modulating flowering related processes. Three of the four MLKs ( MLK1/2/4 ) have been characterized, however, little is known about MLK3 , the most divergent MLKs. Results: We demonstrated that compared with wild type, mlk3 , a truncated MLK3 , flowered slightly early under long day conditions and ectopic expression of MLK3 rescued the morphological defects of mlk3 , indicating that MLK3 negatively regulates flowering. GA 3 application accelerated flowering of both wild type and mlk3 , suggesting that mlk3 had normal GA response. The recombinant MLK3-GFP was localized in the nucleus exclusively. In vitro kinase assay revealed that the nuclear protein MLK3 phosphorylated histone 3 at threonine 3 (H3T3ph). Mutation of a conserved catalytic residue (Lysine 175) abolished the kinase activity and resulted in failure to complement the early flowering phenotype of mlk3 . Interestingly, the global level of H3T3 phosphorylation in mlk3 did not differ significantly from wild type, suggesting the redundant roles of MLKs in flowering regulation. The transcriptomic analysis demonstrated that 425 genes significantly altered expression level in mlk3 relative to wild type. The mlk3 mlk4 double mutant generated by crossing mlk3 with mlk4 , a loss-of-function mutant of MLK4 showing late flowering, flowered between the two parental lines, suggesting that MLK3 played an antagonistic role to MLK4 in plant transition to flowering. Conclusions: A serine/threonine kinase encoding gene MLK3 is a casein kinase 1 specific to the plant species and represses flowering slightly. MLK3 located in nucleus catalyzes the phosphorylation of histone H3 at threonine 3 in vitro and an intact lysine residue (K175) is indispensible for the kinase activity. This study sheds new light on the delicate control of flowering by the plant-specific CK1 in Arabidopsis.

scholarly journals Neuronal Cell Shape and Neurite Initiation Are Regulated by the Ndr Kinase SAX-1, a Member of the Orb6/COT-1/Warts Serine/Threonine Kinase Family

2000 ◽  
Vol 11 (9) ◽  
pp. 3177-3190 ◽  
Author(s):  
Jennifer A. Zallen ◽  
Erin L. Peckol ◽  
David M. Tobin ◽  
Cornelia I. Bargmann
Keyword(s):  
Cell Shape ◽  
Neuronal Cell ◽  
Neurite Growth ◽  
Dominant Negative ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
C Elegans ◽  
Calcium Calmodulin Dependent ◽  
Binding Domains ◽  
Neurite Initiation

The Caenorhabditis elegans sax-1 gene regulates several aspects of neuronal cell shape. sax-1 mutants have expanded cell bodies and ectopic neurites in many classes of neurons, suggesting that SAX-1 functions to restrict cell and neurite growth. The ectopic neurites in sensory neurons of sax-1mutants resemble the defects caused by decreased sensory activity. However, the activity-dependent pathway, mediated in part by the UNC-43 calcium/calmodulin-dependent kinase II, functions in parallel with SAX-1 to suppress neurite initiation. sax-1 encodes a serine/threonine kinase in the Ndr family that is related to the Orb6 (Schizosaccharomyces pombe), Warts/Lats (Drosophila), and COT-1 (Neurospora) kinases that function in cell shape regulation. These kinases have similarity to Rho kinases but lack consensus Rho-binding domains. Dominant negative mutations in the C. elegans RhoA GTPase cause neuronal cell shape defects similar to those ofsax-1 mutants, and genetic interactions betweenrhoA and sax-1 suggest shared functions. These results suggest that SAX-1/Ndr kinases are endogenous inhibitors of neurite initiation and cell spreading.

Ultrastructural Characterization of Platelet-Activating Factor-Stimulated Human Eosinophils from Patients with Asthma

1993 ◽  
Vol 84 (4) ◽  
pp. 391-399 ◽  
Author(s):  
Claus Kroegel ◽  
Ann Dewar ◽  
Tatsuo Yukawa ◽  
Per Venge ◽  
Peter J. Barnes ◽  
...  
Keyword(s):  
Morphometric Analysis ◽  
Morphological Changes ◽  
Shape Change ◽  
Platelet Activating Factor ◽  
Eosinophil Cationic Protein ◽  
Cell Periphery ◽  
Cationic Protein ◽  
Asthmatic Patients ◽  
Transmission Electron

1. Purified human eosinophils from asthmatic patients were stimulated with platelet-activating factor in vitro and examined for morphological changes by transmission electron and light microscopy. Changes were also evaluated by morphometric analysis and were related to the platelet-activating factor-stimulated release of granular eosinophil cationic protein. 2. Stimulation of eosinophils with platelet-activating factor induced a dose-dependent shape change, including the elongation of cells, loss of microvilli and the formation of lamellipodia. This effect was maximal at 25 min and was reversible. 3. Stimulation with platelet-activating factor also induced granule movement to the cell periphery and fusion of adjacent granules. Granules became swollen and vesiculated, whereas both the matrix and core showed evidence of solubilization. 4. There was a time-dependent secretion of eosinophilic cationic protein from human eosinophils upon stimulation with platelet-activating factor which occurred without significant lactate dehydrogenase release. 5. Morphometric analysis of the transmission electron micrographs indicated a significant reduction in cytoplasmic area after 10 min of incubation with platelet-activating factor from 39.0 ± 1.7 μm2 for untreated eosinophils to 33.2 ± 2.3 μm2 (P < 0.02) for platelet-activating factor-treated cells, underscoring the observation that the cells change from spherical to ellipsoidal. No significant increase in the perimeter of the cells was found. 6. The number of granule-profiles in platelet-activating factor-stimulated eosinophils was slightly reduced when compared with control, and an increase in granule area was observed 10 min after platelet-activating factor challenge (0.215 ± 0.011 μm2 versus 0.246 ± 0.016 μm2). 7. Human eosinophils from patients with asthma stimulated with platelet-activating factor undergo both cellular and granular alterations and reorganization which parallel the release of granular eosinophil basic protein.

The Serine/Threonine Kinase Pim-1 Stabilizes 130 Kilodalton FLT3 and Promotes Aberrant Signaling through STAT5 in Acute Myeloid Leukemia Cells with FLT3 Internal Tandem Duplication: Synergistic Apoptosis Induction by Pim-1 and FLT3 Inhibitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 942-942 ◽  
Author(s):  
Yingqiu Xie ◽  
Mehmet Burcu ◽  
Maria R. Baer
Keyword(s):  
Half Life ◽  
Myeloid Leukemia ◽  
Tandem Duplication ◽  
Kinase Activity ◽  
Serine Threonine Kinase ◽  
Internal Tandem Duplication ◽  
Threonine Kinase ◽  
Flt3 Inhibitors ◽  
Flt3 Internal Tandem Duplication

Abstract Abstract 942 Fms-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) results in FLT3 constitutive activation and aberrant signaling in acute myeloid leukemia (AML) cells. FLT3-ITD is associated with adverse treatment outcome in AML, but FLT3 inhibitors have had limited therapeutic efficacy. The oncogenic serine/threonine kinase Pim-1 is upregulated in AML cells with FLT3-ITD. Pim-1 inhibitors are entering clinical trials, and we sought to characterize the role of Pim-1 and the effects of Pim-1 inhibition in FLT3-ITD cells. Wild-type (WT) FLT3 exists predominantly in a 150 kDa complex glycosylated form. In contrast, FLT3-ITD is partially retained in the endoplasmic reticulum (ER) as a misfolded 130 kDa underglycosylated, or high-mannose, species in association with the ER transmembrane chaperone calnexin. In addition, FLT3-ITD also associates with and is stabilized by the cytosolic chaperone heat shock protein (HSP) 90. FLT3-ITD activates signal transducer and activation of transcription (STAT) 5 and upregulates the STAT5 downstream target Pim-1. FLT3 contains a putative Pim-1 substrate consensus serine phosphorylation site, and we hypothesized that FLT3 might be a Pim-1 substrate. FLT3-ITD cell lines studied included MV4-11, MOLM-14 and transfected Ba/F3-ITD, and FLT3 WT cells included BV173, EOL-1 and transfected Ba/F3-WT. Pim-1 activity was measured by an in vitro kinase assay of BAD phosphorylation at serine 112, and Pim-1 expression, FLT3 expression, phosphorylation and co-immunoprecipitation, and STAT5 phosphorylation and expression by Western blot analysis. Pim-1 knockdown was accomplished by infection with lentivirus containing Pim-1 small hairpin RNA (shRNA) or non-target control, and Pim-1 kinase inhibition by incubation with the Pim-1-selective inhibitor quercetagetin. Pim-1 was found to directly interact with and serine-phosphorylate FLT3 from FLT3-ITD, but not FLT3-WT, cells in vitro. Inhibition of Pim-1 kinase disrupted binding of FLT3 to its chaperones calnexin and HSP90, and resulted in decreased expression and half-life of 130 kDa FLT3 and increased expression and half-life of 150 kDa FLT3. The decrease in expression and half-life of 130 kDa FLT3 was partially abrogated by co-incubation with the proteasome inhibitor MG132. Moreover, the increase in 150 Kda FLT3 was abrogated by co-incubation with the glycosylation inhibitor 2-deoxy-D-glucose. Thus Pim-1 maintains FLT3 as a 130 kDa species by enhancing its binding to its chaperones calnexin and HSP90, protecting it from proteasomal degradation and inhibiting its glycosylation to form 150 kDa FLT3. Inhibition of Pim-1 kinase activity also decreased phosphorylation of FLT3 at tyrosine 591, a docking site for binding of FLT3-ITD, but not FLT3-WT, to STAT5, and decreased both STAT5 phosphorylation and expression of Pim-1 itself. In contrast, Pim-1 inhibition had no effect on FLT3 tyrosine kinase activity nor on expression of Pim-2, another Pim kinase family member implicated in promoting survival of FLT3-ITD cells. Finally, the Pim-1 kinase inhibitor quercetagetin and the FLT3 inhibitor PKC412 had a synergistic effect in inducing apoptosis of Ba/F3-ITD cells: We conclude that Pim-1, which is transcriptionally upregulated through STAT5 in FLT3-ITD cells, serine-phosphorylates FLT3-ITD, thereby maintaining it in an underglycosylated form, and promotes STAT5 signaling, and that inhibition of Pim-1 and of FLT3 is synergistic in inducing apoptosis of FLT3-ITD cells. Thus Pim-1 inhibitors should inhibit aberrant signaling upstream as well as downstream of Pim-1 in FLT3-ITD cells, and have the potential to enhance the therapeutic efficacy of FLT3 inhibitors in patients with AML with FLT3-ITD Disclosures: No relevant conflicts of interest to declare.

scholarly journals Protein Serine/Threonine Kinase StkP Positively Controls Virulence and Competence in Streptococcus pneumoniae

2004 ◽  
Vol 72 (4) ◽  
pp. 2434-2437 ◽  
Author(s):  
Jose Echenique ◽  
Aras Kadioglu ◽  
Susana Romao ◽  
Peter W. Andrew ◽  
Marie-Claude Trombe
Keyword(s):  
Streptococcus Pneumoniae ◽  
Lung Infection ◽  
Signaling Network ◽  
Serine Threonine Kinase ◽  
Positive Regulation ◽  
Threonine Kinase

ABSTRACT In the Streptococcus pneumoniae genome, stkP, encoding a membrane-associated serine/threonine kinase, is not redundant (L. Novakova, S. Romao, J. Echenique, P. Branny, and M.-C. Trombe, unpublished results). The data presented here demonstrate that StkP belongs to the signaling network involved in competence triggering in vitro and lung infection and bloodstream invasion in vivo. In competence, functional StkP is required for activation of comCDE upstream of the autoregulated ring orchestrated by the competence-stimulating peptide. This is the first description of positive regulation of comCDE transcription in balance with its repression by CiaRH.

scholarly journals gamma-tubulin is a minus end-specific microtubule binding protein.

1995 ◽  
Vol 131 (1) ◽  
pp. 207-214 ◽  
Author(s):  
Q Li ◽  
H C Joshi
Keyword(s):  
Chromosome Segregation ◽  
Structural Integrity ◽  
Cell Periphery ◽  
Vitro Assay ◽  
Interphase Cells ◽  
Gamma Tubulin ◽  
Slow Growing ◽  
Directional Transport

The role of microtubules in mediating chromosome segregation during mitosis is well-recognized. In addition, interphase cells depend upon a radial and uniform orientation of microtubules, which are intrinsically asymmetric polymers, for the directional transport of many cytoplasmic components and for the maintenance of the structural integrity of certain organelles. The slow growing minus ends of microtubules are linked to the centrosome ensuring extension of the fast growing plus ends toward the cell periphery. However, the molecular mechanism of this linkage is not clear. One hypothesis is that gamma-tubulin, located at the centrosome, binds to the minus ends of microtubules. To test this model, we synthesized radiolabeled gamma-tubulin in vitro. We demonstrate here biochemically a specific, saturable, and tight (Kd = 10(-10) M) interaction of gamma-tubulin and microtubule ends with a stoichiometry of 12.6 +/- 4.9 molecules of gamma-tubulin per microtubule. In addition, we designed an in vitro assay to visualize gamma-tubulin at the minus ends of axonemal microtubules. These data show that gamma-tubulin represents the first protein to bind microtubule minus ends and might be responsible for mediating the link between microtubules and the centrosome.

Sign in / Sign up

Export Citation Format

Share Document