Regulation of the Actin Cytoskeleton Organization in Yeast by a Novel Serine/Threonine Kinase Prk1p

Normal actin cytoskeleton organization in budding yeast requires the function of the Pan1p/ End3p complex. Mutations in PAN1 and END3 cause defects in the organization of actin cytoskeleton and endocytosis. By screening for mutations that can suppress the temperature sensitivity of a pan1 mutant (pan1-4), a novel serine/threonine kinase Prk1p is now identified as a new factor regulating the actin cytoskeleton organization in yeast. The suppression of pan1-4 by prk1 requires the presence of mutant Pan1p. Although viable, the prk1 mutant is unable to maintain an asymmetric distribution of the actin cytoskeleton at 37°C. Consistent with its role in the regulation of actin cytoskeleton, Prk1p localizes to the regions of cell growth and coincides with the polarized actin patches. Overexpression of the PRK1 gene in wild-type cells leads to lethality and actin cytoskeleton abnormalities similar to those exhibited by the pan1 and end3 mutants. In vitro phosphorylation assays demonstrate that Prk1p is able to phosphorylate regions of Pan1p containing the LxxQxTG repeats, including the region responsible for binding to End3p. Based on these findings, we propose that the Prk1 protein kinase regulates the actin cytoskeleton organization by modulating the activities of some actin cytoskeleton-related proteins such as Pan1p/End3p.

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Regulation of Yeast Actin Cytoskeleton-Regulatory Complex Pan1p/Sla1p/End3p by Serine/Threonine Kinase Prk1p

Molecular Biology of the Cell ◽

10.1091/mbc.12.12.3759 ◽

2001 ◽

Vol 12 (12) ◽

pp. 3759-3772 ◽

Cited By ~ 72

Author(s):

Guisheng Zeng ◽

Xianwen Yu ◽

Mingjie Cai

Keyword(s):

Actin Cytoskeleton ◽

Regulatory Protein ◽

Strong Support ◽

Stable Complex ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization

The serine/threonine kinase Prk1p is known to be involved in the regulation of the actin cytoskeleton organization in budding yeast. One possible function of Prk1p is the negative regulation of Pan1p, an actin patch regulatory protein that forms a complex in vivo with at least two other proteins, Sla1p and End3p. In this report, we identified Sla1p as another substrate for Prk1p. The phosphorylation of Sla1p by Prk1p was established in vitro with the use of immunoprecipitated Prk1p and in vivo with the use ofPRK1 overexpression, and was further supported by the finding that immunoprecipitated Sla1p contained PRK1- and ARK1-dependent kinase activities. Stable complex formation between Prk1p and Sla1p/Pan1p in vivo could be observed once the phosphorylation reaction was blocked by mutation in the catalytic site of Prk1p. Elevation of Prk1p activities in wild-type cells resulted in a number of deficiencies, including those in colocalization of Pan1p and Sla1p, endocytosis, and cell wall morphogenesis, likely attributable to a disintegration of the Pan1p/Sla1p/End3p complex. These results lend a strong support to the model that the phosphorylation of the Pan1p/Sla1p/End3p complex by Prk1p is one of the important mechanisms by which the organization and functions of the actin cytoskeleton are regulated.

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

10.21203/rs.2.16145/v1 ◽

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.

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Inhibition of Bcr serine kinase by tyrosine phosphorylation.

Molecular and Cellular Biology ◽

10.1128/mcb.16.3.998 ◽

1996 ◽

Vol 16 (3) ◽

pp. 998-1005 ◽

Cited By ~ 29

Author(s):

J Liu ◽

Y Wu ◽

G Z Ma ◽

D Lu ◽

L Haataja ◽

...

Keyword(s):

Protein Kinase ◽

Tyrosine Phosphorylation ◽

Kinase Activity ◽

Philadelphia Chromosome ◽

Wild Type ◽

Serine Kinase ◽

Serine Threonine Kinase ◽

Threonine Kinase

The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired.

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A SAC phosphoinositide phosphatase controls rice development via hydrolyzing phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate

10.1101/740001 ◽

2019 ◽

Author(s):

Tao Guo ◽

Hua-Chang Chen ◽

Zi-Qi Lu ◽

Min Diao ◽

Ke Chen ◽

...

Keyword(s):

Actin Polymerization ◽

Membrane Lipids ◽

Dependent Manner ◽

Cytoskeleton Organization ◽

Cellular Processes ◽

Phosphoinositide Phosphatase ◽

Actin Cytoskeleton Organization ◽

Phosphatidylinositol 4 ◽

Related Proteins

AbstractPhosphoinositides (PIs) as regulatory membrane lipids play essential roles in multiple cellular processes. Although the exact molecular targets of PIs-dependent modulation remain largely elusive, the effects of disturbed PIs metabolism could be employed to propose regulatory modules associated with particular downstream targets of PIs. Here, we identified the role of GRAIN NUMBER AND PLANT HEIGHT 1 (GH1), which encodes a suppressor of actin (SAC) domain-containing phosphatase with unknown function in rice. Endoplasmic reticulum-localized GH1 specifically dephosphorylated and hydrolyzed phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Inactivation of GH1 resulted in massive accumulation of both PI4P and PI(4,5)P2, while excessive GH1 caused their depletion. Notably, superabundant PI4P and PI(4,5)P2 could both disrupt actin cytoskeleton organization and suppress cell elongation. Interestingly, both PI4P and PI(4,5)P2 inhibited actin-related proteins 2 and 3 (Arp2/3) complex-nucleated actin branching networks in vitro, whereas PI(4,5)P2 showed more dramatic effect in a dose-dependent manner. Overall, the overaccumulation of PI(4,5)P2 resulted from dysfunction of SAC phosphatase possibly perturbs Arp2/3 complex-mediated actin polymerization, thereby disordering the cell development. These findings imply that Arp2/3 complex might be the potential molecular target of PI(4,5)P2-dependent modulation in eukaryotes, thereby providing new insights into the relationship between PIs homeostasis and plants growth and development.

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MiRNA-155 targets myosin light chain kinase and modulates actin cytoskeleton organization in endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00521.2013 ◽

2014 ◽

Vol 306 (8) ◽

pp. H1192-H1203 ◽

Cited By ~ 43

Author(s):

Martina Weber ◽

Sinae Kim ◽

Nicole Patterson ◽

Kimberly Rooney ◽

Charles D. Searles

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Actin Cytoskeleton ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization ◽

Mouse Aorta

Previously, we identified a microRNA (miRNA) signature for endothelial cells (ECs) subjected to unidirectional shear stress (USS). MiR-155, a multifunctional miRNA that has been implicated in atherosclerosis, was among the shear stress-responsive miRNAs. Here, we examined the role of miR-155 in modulating EC phenotype and function. In vitro, increased miR-155 levels in human ECs induced changes in morphology and filamentous (F)-actin organization. In addition, ECs transfected with miR-155 mimic were less migratory and less proliferative and had less apoptosis compared with control ECs. In mouse aorta, miR-155 expression was increased in the intima of thoracic aorta, where blood flow produces steady and unidirectional shear stress, compared with the intima of the lower curvature of the aortic arch, which is associated with oscillatory and low shear stress. These differences in miR-155 expression were associated with distinct changes in EC morphology and F-actin. The effects of miR-155 in vitro were mediated through suppression of two key regulators of the EC cytoskeleton organization: RhoA and myosin light chain kinase (MYLK). A novel direct interaction between miR-155 and the MYLK 3′UTR was verified by luciferase-MYLK 3′UTR reporter assays. Furthermore, the intensity of immunofluorescence staining for RhoA and MYLK in mouse aorta correlated inversely with miR-155 expression. In conclusion, a prominent effect of the multifunctional miR-155 in ECs is modulation of phenotype through alterations in RhoA, MYLK expression, and actin cytoskeleton organization.

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Inhibition of ULK2 Autophagic Activity by Its Association with YAP

Journal of Biology and Life Science ◽

10.5296/jbls.v8i1.10214 ◽

2016 ◽

Vol 8 (1) ◽

pp. 12 ◽

Cited By ~ 1

Author(s):

Sung Hwa Shin ◽

Eun Jeoung Lee ◽

Sunghee Hyun ◽

Dowonkyoung Park ◽

Sang Sun Kang

Keyword(s):

Mammalian Cells ◽

Signal Transduction Pathway ◽

Transduction Pathway ◽

Wild Type ◽

Therapeutic Approaches ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Py Motif

Uncoordinated 51-like kinase 2 (ULK2) is a member of the serine/threonine kinase family that functions an essential role regulating autophagy in mammalian cells. As autophagy is implicated in normal cellular homeostasis and multiple diseases, better mechanisticinsight will drive thedevelopment of novel therapeutic approaches. Here, we present evidence that ULK2 interacts withYAP for its degradation and subcellular localization. A potential PPxY motif (328PPnY331) was identified, which is similar with the consensus PPxY motif in ULK2 S/P domain. TheP329A (PA) mutation in the PY motif of ULK2 abolished the YAP-ULK2 association.At first, we observed that ULK2 physically interacted to YAP in vivo and in vitro, using a pull-down approach. Secondly, ULK2 and YAP co- localized at the apical (or tight junction) membrane as visualized by confocal microscopy. Furthermore, the PA mutant substantially increased during autophagy than that of wild-type ULK2 or the P242A mutant in transient transfection assays. Thus, the association between ULK2 and YAP through the WW domain links autophagy and the Hippo signal transduction pathway.

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SH3 domain-containing proteins and the actin cytoskeleton in yeast

Biochemical Society Transactions ◽

10.1042/bst0331247 ◽

2005 ◽

Vol 33 (6) ◽

pp. 1247-1249 ◽

Cited By ~ 6

Author(s):

G. Mirey ◽

A. Soulard ◽

C. Orange ◽

S. Friant ◽

B. Winsor

Keyword(s):

Actin Cytoskeleton ◽

Protein Interactions ◽

Membrane Trafficking ◽

Actin Polymerization ◽

Protein Protein Interactions ◽

Yeast Saccharomyces Cerevisiae ◽

Sh3 Domains ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization

SH3 (Src homology-3) domains are involved in protein–protein interactions through proline-rich domains. Many SH3-containing proteins are implicated in actin cytoskeleton organization. The aim of our ongoing work is to study the functions of the SH3-containing proteins in actin cytoskeleton regulation. The yeast Saccharomyces cerevisiae proteome includes 29 SH3 domains distributed in 25 proteins. We have examined the direct involvement of these SH3 domains in actin polymerization using an in vitro polymerization assay on GST (glutathione S-transferase)–SH3-coated beads. As expected, not all SH3 domains show polymerization activity, and many recruit distinct partners as assessed by microscopy and pull-down experiments. One such partner, Las17p, the yeast homologue of WASP (Wiskott–Aldrich syndrome protein), was assayed because it stimulates actin nucleation via the Arp2/3 (actin-related protein 2/3) complex. Ultimately, proteins involved in specific biological processes, such as membrane trafficking, may also be recruited by some of these SH3 domains, shedding light on the SH3-containing proteins and actin cytoskeleton functions in these processes.

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The actin cytoskeleton organization and disorganization properties of the photosynthetic dinoflagellate Symbiodinium kawagutii in culture

Canadian Journal of Microbiology ◽

10.1139/cjm-2014-0325 ◽

2014 ◽

Vol 60 (11) ◽

pp. 767-775 ◽

Cited By ~ 4

Author(s):

Marco A. Villanueva ◽

Georgina Arzápalo-Castañeda ◽

Raúl Eduardo Castillo-Medina

Keyword(s):

Actin Cytoskeleton ◽

Freeze Fracture ◽

Fluorescein Isothiocyanate ◽

Cytoskeleton Organization ◽

Reticular Pattern ◽

Thick Filaments ◽

Actin Cytoskeleton Organization ◽

Butanedione Monoxime ◽

Marine Dinoflagellates

The actin cytoskeleton organization in symbiotic marine dinoflagellates is largely undescribed; most likely, due to their intense pigment autofluorescence and cell walls that block fluorescent probe access. Using a freeze–fracture and fixation procedure, we observed the actin cytoskeleton of Symbiodinium kawagutii cultured in vitro with fluorescently labeled phalloidin and by indirect immunofluorescence with monoclonal antibodies specific for actin. The cytoskeleton appeared as an organized network with interconnected cortical and cytoplasmic thick filaments, along with some intertwined fine filaments. It showed a grid-type, reticular pattern organized in a lattice-like structure within the cell and throughout the cytoplasm. This organization was similar when the observations were done with either fluorescein isothiocyanate (FITC)–phalloidin or anti-actin, although the latter showed a more evenly distributed fluorescence characteristic of nonpolymerized actin. The network organization collapsed upon treatment with latrunculin, resulting in bright foci and diffuse fluorescence. A similar effect was obtained upon butanedione monoxime treatment, except that no bright foci were observed. We have been able to successfully visualize the actin cytoskeleton of S. kawagutii cells using fluorescence-based procedures. This is the first report on the visualization of the organization of the actin cytoskeleton under various conditions in these walled, highly autofluorescent cells.

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

Biochemical Journal ◽

10.1042/bj20080234 ◽

2008 ◽

Vol 415 (1) ◽

pp. 27-33 ◽

Cited By ~ 45

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.

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