scholarly journals Transmembrane Signaling Regulates the Remodeling of the Actin Cytoskeleton:Roles of PKC and Adducin

2021 ◽  
Author(s):  
Bih-Hwa Shieh ◽  
Wesley Sun ◽  
Darwin Ferng
Keyword(s):  
Actin Cytoskeleton ◽  
Null Allele ◽  
Dominant Negative ◽  
Visual Signaling ◽  
Kinase C ◽  
Severe Effect ◽  
Cross Links ◽  
Dominant Negative Activity ◽  
Spectrin Network

Members of the conventional protein kinase C (cPKC) family are activated by both DAG and Ca2+ and have been implicated in the regulation of the actin cytoskeleton. Drosophila contains two cPKCs, Pkc53E (Pkc1) and eye-PKC (Pkc2); mutants missing each PKC lead to retinal degeneration. While eye-PKC is critical for the visual signaling, the role of Pkc53E is not known. We identified a photoreceptor-specific isoform of Pkc53E and show Pkc53E-RNAi negatively impacts the actin cytoskeleton of rhabdomeres. Interestingly, Pkc53E-RNAi enhances the degeneration of norpAP24 photoreceptors, suggesting Pkc53E could be activated independently of NorpA/PLCβ4. We further demonstrate that in norpAP24 photoreceptors Plc21C can be activated by Gq, which is responsible for the activation of Pkc53E. We explored whether Pkc53E regulates adducin in Drosophila photoreceptors. Adducin cross-links the actin cytoskeleton to the spectrin network, which is blunted by PKC phosphorylation. Importantly, we observed that phosphorylation of adducin was greatly reduced in a null allele of pkc53E. Downregulation of hts that encodes Drosophila adducin, exerts a more severe effect than Pkc53E-RNAi to impact the actin cytoskeleton. In contrast, overexpression of a mCherry tagged Add2, one of the three Drosophila adducin isoforms, led to the apical expansion of rhabdomeres with overgrowth of the actin cytoskeleton. This phenotype is likely caused by the dominant-negative activity of the tagged Add2 as it also was observed in α-spectrin-RNAi or β-spectrin-RNAi. Interestingly, downregulation of Pkc53E does not suppress the expansion of rhabdomeres during development, but negatively affects the appearance of rhabdomeres in adult photoreceptors. We conclude that Drosophila adducin has two distinct functions: in pupal photoreceptors, it regulates rhabdomere morphogenesis, which is independent of Pkc53E. In adult photoreceptors, it promotes the maintenance of the actin cytoskeleton, which is regulated by Pkc53E in response to the light-induced activation of the PLCβ activity.

Role of PKC isoforms in glucose transport in 3T3-L1 adipocytes: insignificance of atypical PKC

2002 ◽  
Vol 283 (2) ◽  
pp. E338-E345 ◽  
Author(s):  
Masatoshi Tsuru ◽  
Hideki Katagiri ◽  
Tomoichiro Asano ◽  
Tetsuya Yamada ◽  
Shigeo Ohno ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Dominant Negative ◽  
Transport Activity ◽  
Wild Type ◽  
Atypical Pkc ◽  
Endogenous Expression ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Pkc Ζ

To elucidate the involvement of protein kinase C (PKC) isoforms in insulin-induced and phorbol ester-induced glucose transport, we expressed several PKC isoforms, conventional PKC-α, novel PKC-δ, and atypical PKC isoforms of PKC-λ and PKC-ζ, and their mutants in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Endogenous expression and the activities of PKC-α and PKC-λ/ζ, but not of PKC-δ, were detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC isoform induced a large amount of PKC activity in 3T3-L1 adipocytes. Phorbol 12-myristrate 13-acetate (PMA) activated PKC-α and exogenous PKC-δ but not atypical PKC-λ/ζ. Insulin also activated the overexpressed PKC-δ but not PKC-α. Expression of the wild-type PKC-α or PKC-δ resulted in significant increases in glucose transport activity in the basal and PMA-stimulated states. Dominant-negative PKC-α expression, which inhibited the PMA activation of PKC-α, decreased in PMA-stimulated glucose transport. Glucose transport activity in the insulin-stimulated state was increased by the expression of PKC-δ but not of PKC-α. These findings demonstrate that both conventional and novel PKC isoforms are involved in PMA-stimulated glucose transport and that other novel PKC isoforms could participate in PMA-stimulated and insulin-stimulated glucose transport. Atypical PKC-λ/ζ was not significantly activated by insulin, and expression of the wild-type, constitutively active, and dominant-negative mutants of atypical PKC did not affect either basal or insulin-stimulated glucose transport. Thus atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.

scholarly journals Autophagy and protein kinase C are required for cardioprotection by sulfaphenazole

2010 ◽  
Vol 298 (2) ◽  
pp. H570-H579 ◽  
Author(s):  
Chengqun Huang ◽  
Wayne Liu ◽  
Cynthia N. Perry ◽  
Smadar Yitzhaki ◽  
Youngil Lee ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Enhanced Recovery ◽  
Ischemia Reperfusion ◽  
Dominant Negative ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Kinase C ◽  
Rat Hearts

Previously, we showed that sulfaphenazole (SUL), an antimicrobial agent that is a potent inhibitor of cytochrome P4502C9, is protective against ischemia-reperfusion (I/R) injury (Ref. 15 ). The mechanism, however, underlying this cardioprotection, is largely unknown. With evidence that activation of autophagy is protective against simulated I/R in HL-1 cells, and evidence that autophagy is upregulated in preconditioned hearts, we hypothesized that SUL-mediated cardioprotection might resemble ischemic preconditioning with respect to activation of protein kinase C and autophagy. We used the Langendorff model of global ischemia to assess the role of autophagy and protein kinase C in myocardial protection by SUL during I/R. We show that SUL enhanced recovery of function, reduced creatine kinase release, decreased infarct size, and induced autophagy. SUL also triggered PKC translocation, whereas inhibition of PKC with chelerythrine blocked the activation of autophagy in adult rat cardiomyocytes. In the Langendorff model, chelerythrine suppressed autophagy and abolished the protection mediated by SUL. SUL increased autophagy in adult rat cardiomyocytes infected with GFP-LC3 adenovirus, in isolated perfused rat hearts, and in mCherry-LC3 transgenic mice. To establish the role of autophagy in cardioprotection, we used the cell-permeable dominant-negative inhibitor of autophagy, Tat-Atg5K130R. Autophagy and cardioprotection were abolished in rat hearts perfused with recombinant Tat-Atg5K130R. Taken together, these studies indicate that cardioprotection mediated by SUL involves a PKC-dependent induction of autophagy. The findings suggest that autophagy may be a fundamental process that enhances the heart's tolerance to ischemia.

Cross-Talk between Rho GTPases Regulates Actin Cytoskeleton and Chemotaxis of Hematopoietic Progenitor Cell.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 266-266
Author(s):  
Hee-Don Chae ◽  
Katherine E. Lee ◽  
Aparna C. Jasti ◽  
David A. Williams ◽  
Yi Gu
Keyword(s):  
Cell Membrane ◽  
Actin Cytoskeleton ◽  
Cross Talk ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Dominant Negative ◽  
Actin Assembly ◽  
Hematopoietic Progenitor ◽  
Stimulation Of

Abstract Movement of hematopoietic stem/progenitor cells into (engraftment) and out of (mobilization) the bone marrow involves actin cytoskeleton and chemotaxis. Members of the Rho GTPase family have been well known for their critical roles in morphogenesis and cell migration via regulating actin assembly. Loss of Rac1 and Rac2 alleles leads to defective engraftment and massive mobilization of hematopoietic progenitor cells (HPCs), which are associated with impaired chemotaxis and cortical filamentous (F)-actin polymerization (Gu et al., Science 302: 445–449). RhoH, a hematopoietic-specific member of the RhoE subfamily, negatively regulates HPC engraftment, chemotaxis, F-actin polymerization and Rac activities (Gu et al., Blood 105: 1467–1475). These findings suggest that RhoH may antagonize Rac function in regulating these cellular processes. However, molecular mechanism of the cross-talk between these Rho GTPases is not defined. In this study, we examined the role of RhoH in actin cytoskeleton organization, chemotaxis and Rac membrane translocation in response to stromal-derived factor 1α (SDF-1α) using RhoH-deficient HPCs and retrovirus-mediated expression of EGFP-fusion proteins. RhoH−/− HPCs exhibit increased migration in response to SDF-1α, especially at low concentration, as compared with wild-type (WT) cells [10ng/ml SDF-1α: 3.5 +/− 0.9 vs. 12.3 +/− 1.8; 100ng/ml SDF-1α: 21.4 +/− 1.7 vs. 32.3 +/− 3.4, migrated cells (%), WT vs. RhoH−/−, n=3, p< 0.01]. Migration without SDF-1α stimulation of RhoH−/− cells is also enhanced. RhoH−/− HPCs assemble cortical F-actin without SDF-1α stimulation, under conditions in which WT cells do not show F-actin polymerization [cells with F-actin (%): 8.9 +/− 0.9 vs. 72.8 +/− 4, WT vs. RhoH−/−, n=6, p<0.001]. Additionally, RhoH−/− HPCs exhibit increased active, GTP-bound Rac GTPases. PAK, a known downstream effector of Rac in regulating actin cytoskeleton, also shows hyperphosphorylation in RhoH-/− HPCs, suggesting that RhoH may regulate actin assembly and cell migration through Rac-mediated pathway. In support of this, expression of a dominant negative Rac1N17 mutant blocks cortical F-actin assembly in RhoH−/− cells [cells with F-actin (%): 60 +/− 1 vs. 19 +/− 7, EGFP-Rac1 vs. Rac1N17, n=2]. To further address the mechanism by which RhoH cross-talks to affect Rac signaling, we examine the role of RhoH in subcellular localization of EGFP-Rac proteins. SDF-1α induces activation of Rac, leading to translocation to the cell membrane where it co-localizes with lipid rafts and mediates cortical F-actin assembly in HPCs. In contrast, the dominant negative Rac1N17 does not localize to the cell membrane after SDF-1α stimulation. In RhoH−/− HPCs, EGFP-Rac protein presents at the cell membrane in the absence of SDF-1α [cells with membrane-localized EGFP-Rac1 (%): 7.5 +/− 3.9 vs. 44.5 +/− 6.4, WT vs. RhoH−/−, n=2]. In contrast, overexpression of RhoH in HPCs blocks translocation to the cell membrane after SDF-1α stimulation of Rac1, Rac2 and active Rac1V12. Finally, we found that RhoH, a constitutively active, GTP-bound protein, preferentially localizes to the cell membrane even in the absence of SDF-1α. This localization is dependent upon the prenylation site and the c-terminal domains of RhoH. Lack of membrane localization is associated with defective biological function. Together, our data suggest that RhoH is essential for proper cortical F-actin assembly and chemotaxis of HPCs via regulating Rac activation and membrane localization, and implicates a functional cross-talk between RhoH and Rac.

p21-Activated kinases regulate actin remodeling in glomerular podocytes

2010 ◽  
Vol 298 (4) ◽  
pp. F951-F961 ◽  
Author(s):  
Jianxin Zhu ◽  
Ortal Attias ◽  
Lamine Aoudjit ◽  
Ruihua Jiang ◽  
Hiroshi Kawachi ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Tyrosine Phosphorylation ◽  
Adaptor Proteins ◽  
Dominant Negative ◽  
Puromycin Aminonucleoside ◽  
Actin Remodeling ◽  
Cellular Processes ◽  
Aminonucleoside Nephrosis ◽  
Puromycin Aminonucleoside Nephrosis

The tyrosine phosphorylation of nephrin is reported to regulate podocyte morphology via the Nck adaptor proteins. The Pak family of kinases are regulators of the actin cytoskeleton and are recruited to the plasma membrane via Nck. Here, we investigated the role of Pak in podocyte morphology. Pak1/2 were expressed in cultured podocytes. In mouse podocytes, Pak2 was predominantly phosphorylated, concentrated at the tips of the cellular processes, and its expression and/or phosphorylation were further increased when differentiated. Overexpression of rat nephrin in podocytes increased Pak1/2 phosphorylation, which was abolished when the Nck binding sites were mutated. Furthermore, dominant-negative Nck constructs blocked the Pak1 phosphorylation induced by antibody-mediated cross linking of nephrin. Transient transfection of constitutively kinase-active Pak1 into differentiated mouse podocytes decreased stress fibers, increased cortical F-actin, and extended the cellular processes, whereas kinase-dead mutant, kinase inhibitory construct, and Pak2 knockdown by shRNA had the opposite effect. In a rat model of puromycin aminonucleoside nephrosis, Pak1/2 phosphorylation was decreased in glomeruli, concomitantly with a decrease of nephrin tyrosine phosphorylation. These results suggest that Pak contributes to remodeling of the actin cytoskeleton in podocytes. Disturbed nephrin-Nck-Pak interaction may contribute to abnormal morphology of podocytes and proteinuria.

scholarly journals Phosphorylated Pleckstrin Induces Cell Spreading via an Integrin-Dependent Pathway

2000 ◽  
Vol 150 (6) ◽  
pp. 1461-1466 ◽  
Author(s):  
Richard L. Roll ◽  
Eve Marie Bauman ◽  
Joel S. Bennett ◽  
Charles S. Abrams
Keyword(s):  
Chimeric Protein ◽  
Cytoplasmic Tail ◽  
Cell Spreading ◽  
Competitive Inhibitor ◽  
Dominant Negative ◽  
Kinase C ◽  
Dominant Negative Inhibitor ◽  
Β Chain ◽  
Dependent Pathway

Pleckstrin is a 40-kD phosphoprotein containing NH2- and COOH-terminal pleckstrin homology (PH) domains separated by a disheveled-egl 10-pleckstrin (DEP) domain. After platelet activation, pleckstrin is rapidly phosphorylated by protein kinase C. We reported previously that expressed phosphorylated pleckstrin induces cytoskeletal reorganization and localizes in microvilli along with glycoproteins, such as integrins. Given the role of integrins in cytoskeletal organization and cell spreading, we investigated whether signaling from pleckstrin cooperated with signaling pathways involving the platelet integrin, αIIbβ3. Pleckstrin induced cell spreading in both transformed (COS-1 & CHO) and nontransformed (REF52) cell lines, and this spreading was regulated by pleckstrin phosphorylation. In REF52 cells, pleckstrin-induced spreading was matrix dependent, as evidenced by spreading of these cells on fibrinogen but not on fibronectin. Coexpression with αIIbβ3 did not enhance pleckstrin-mediated cell spreading in either REF52 or CHO cells. However, coexpression of the inactive variant αIIbβ3 Ser753Pro, or β3 Ser753Pro alone, completely blocked pleckstrin-induced spreading. This implies that αIIbβ3 Ser753Pro functions as a competitive inhibitor by blocking the effects of an endogenous receptor that is used in the signaling pathway involved in pleckstrin-induced cell spreading. Expression of a chimeric protein composed of the extracellular and transmembrane portion of Tac fused to the cytoplasmic tail of β3 completely blocked pleckstrin-mediated spreading, whereas chimeras containing the cytoplasmic tail of β3 Ser753Pro or αIIb had no effect. This suggests that the association of an unknown signaling protein with the cytoplasmic tail of an endogenous integrin β-chain is also required for pleckstrin-induced spreading. Thus, expressed phosphorylated pleckstrin promotes cell spreading that is both matrix and integrin dependent. To our knowledge, this is the first example of a mutated integrin functioning as a dominant negative inhibitor.

scholarly journals Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C.

1992 ◽  
Vol 116 (3) ◽  
pp. 695-706 ◽  
Author(s):  
G P Downey ◽  
C K Chan ◽  
P Lea ◽  
A Takai ◽  
S Grinstein
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Protein Phosphorylation ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Actin Assembly ◽  
Membrane Ruffling ◽  
Kinase C

The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified. A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC. It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils. Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBD-phallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin. [32P]ATP was used to detect protein phosphorylation in electroporated cells. Exposure of neutrophils to 4 beta-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the actin cytoskeleton, whereas the 4a-isomer, which is inactive towards PKC, failed to produce any of these changes. Moreover, 1,2-dioctanoylglycerol, mezerein, and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, which are nonphorbol activators of PKC, also promoted actin assembly. Although these effects were consistent with a role of PKC, the following observations suggested that stimulation of conventional isoforms of the kinase were not directly responsible for actin assembly: (a) Okadaic acid, an inhibitor of phosphatases 1 and 2A, potentiated PMA-induced protein phosphorylation, but not actin assembly; and (b) PMA-induced actin assembly and membrane ruffling were not prevented by the conventional PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, staurosporine, calphostin C, or sphingosine at concentrations that precluded PMA-induced protein phosphorylation and superoxide production. On the other hand, PMA-induced actin assembly was inhibited by long-chain fatty acid coenzyme A esters, known inhibitors of nuclear PKC (nPKC). We conclude that PMA-induced actin assembly is unlikely to be mediated by the conventional isoforms of PKC, but may be mediated by novel isoforms of the kinase such as nPKC.

scholarly journals Evidence That Atypical Protein Kinase C-λ and Atypical Protein Kinase C-ζ Participate in Ras-mediated Reorganization of the F-actin Cytoskeleton

1999 ◽  
Vol 144 (3) ◽  
pp. 413-425 ◽  
Author(s):  
Florian Überall ◽  
Karina Hellbert ◽  
Sonja Kampfer ◽  
Karl Maly ◽  
Andreas Villunger ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Dominant Negative ◽  
Stress Fibers ◽  
Atypical Protein Kinase C ◽  
Nih3t3 Cells ◽  
Atypical Protein Kinase ◽  
Kinase C ◽  
Actin Stress Fibers

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes λ and ζ, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-λ and aPKC-ζ, as well as antisense constructs encoding RNA-directed against isotype-specific 5′ sequences of the corresponding mRNA, abrogates the Ha-Ras–induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-α was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-λ and aPKC-ζ lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3′-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-λ acts upstream of PI3K and Rac-1, whereas aPKC-ζ functions downstream of PI3K and Rac-1. This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-λ or aPKC-ζ results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-ζ was abrogated by coexpression of DN Rac-1 N17.

scholarly journals Kinase-Inactivated ULK Proteins Inhibit Autophagy via Their Conserved C-Terminal Domains Using an Atg13-Independent Mechanism

2008 ◽  
Vol 29 (1) ◽  
pp. 157-171 ◽  
Author(s):  
Edmond Y. W. Chan ◽  
Andrea Longatti ◽  
Nicole C. McKnight ◽  
Sharon A. Tooze
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Intramolecular Interactions ◽  
Regulatory Function ◽  
Independent Mechanism ◽  
Dominant Negative Activity

ABSTRACT The yeast Atg1 serine/threonine protein kinase and its mammalian homologs ULK1 and ULK2 play critical roles during the activation of autophagy. Previous studies have demonstrated that the conserved C-terminal domain (CTD) of ULK1 controls the regulatory function and localization of the protein. Here, we explored the role of kinase activity and intramolecular interactions to further understand ULK function. We demonstrate that the dominant-negative activity of kinase-dead mutants requires a 7-residue motif within the CTD. Our data lead to a model in which the functions of ULK1 and ULK2 are controlled by autophosphorylation and conformational changes involving exposure of the CTD. Additional mapping indicates that the CTD contains other distinct regions that direct membrane association and interaction with the putative human homologue of Atg13, which we have here characterized. Atg13 is required for autophagy and Atg9 trafficking during autophagy. However, Atg13 does not bind the 7-residue dominant-negative motif in the CTD of ULK proteins nor is the inhibitory activity of the CTDs rescued by Atg13 ectopic expression, suggesting that in mammalian cells, the CTD may interact with additional autophagy proteins.

Sign in / Sign up

Export Citation Format

Share Document