Activation of SGK1 by HGF, Rac1 and integrin-mediated cell adhesion in MDCK cells: PI-3K-dependent and -independent pathways

2002 ◽  
Vol 115 (9) ◽  
pp. 1985-1993 ◽  
Author(s):  
Candace Shelly ◽  
Roman Herrera
Keyword(s):  
Transcription Factor ◽  
Cell Survival ◽  
Binding Proteins ◽  
Mdck Cells ◽  
Phosphorylation Site ◽  
Dominant Negative ◽  
Hydrophobic Domain ◽  
Akt Phosphorylation ◽  
Gtp Binding Proteins ◽  
Gtp Binding

The SGK1 protein belongs to the AGC gene family of kinases that are regulated by phosphorylation mediated by PDK1. SGK1 regulation is accomplished by several pathways including growth-factor and stress-mediated signaling. We have expanded the analysis of SGK1 regulation in epithelial cells. We used HA-tagged SGK1 to transiently transfect MDCK cells and study the regulation of SGK1 upon stimulation with HGF, cAMP or upon adhesion of the cells to immobilized fibronectin. In addition, we studied the regulation of SGK1 activity by small GTP-binding proteins of the Rho family. Treatment of MDCK cells with HGF leads to a time-dependent activation of SGK1 that is blocked by wortmanin. This activation requires the conserved phosphorylation site present in the activation loop of the kinase (T256 in SGK1) and the phosphorylation site present in a hydrophobic domain at its C-terminus (S422 in SGK1), which are targets for PDK1/PDK2-mediated regulation of SGK1. We tested whether SGK1 could be activated by cAMP as it contains a putative PKA site. We were unable to demonstrate a significant activation of HA-SGK1 by cAMP stimulation under conditions where we detect cAMP-mediated phosphorylation of the transcription factor CREB. Cotransfection of SGK1 with activated small GTP-binding proteins revealed that Rac1, but not Rho or Rap1, induces activation of SGK1. However, this activation was wortmanin insensitive and dominant-negative Rac1 did not inhibit the HGF-mediated activation of SGK1. Adhesion of MDCK cells to immobilized fibronectin also leads to activation of SGK1. However, it appears that the integrin-mediated activation of HA-SGK1 differs from AKT activation in the fact that AKT phosphorylation was blocked by wortmanin (or LY294002)whereas HA-SGK1 was not. The adhesion-dependent activation, however, requires the intact phosphorylation sites of SGK1. Co-transfection of HA-SGK1 with RacV12 results in increased activity in adherent cells compared with HA-SGK1 alone. Since RacN17 failed to inhibit adhesion dependent-activation of SGK1,it suggests that integrin activation is achieved by a parallel Rac-independent pathway. The activation of SGK1 by HGF and integrin provides a link between HGF-mediated protection of MDCK from de-attachment induced apoptosis(anoikis). We demonstrate that dephosphorylation of the transcription factor FKRHL1 induced by cell de-attachment is prevented by activated SGK1,suggesting that SGK1 regulates cell survival pathways. In summary, we demonstrate that SGK1 activation could be achieved through signaling pathways involved in the regulation of cell survival, cell-cell and cell-matrix interactions. SGK1 activation can be accomplished via HGF,PI-3K-dependent pathways and by integrin-mediated, PI-3K independent pathways. In addition, activation of SGK1 by the small GTP-binding protein Rac1 has been observed.

Role of small GTP binding proteins in the growth-promoting and antiapoptotic actions of gastrin

2004 ◽  
Vol 287 (3) ◽  
pp. G715-G725 ◽  
Author(s):  
Vinzenz Stepan ◽  
Saravanan Ramamoorthy ◽  
Nonthalee Pausawasdi ◽  
Craig D. Logsdon ◽  
Frederick K. Askari ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Binding Proteins ◽  
Dominant Negative ◽  
Growth And Survival ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Growth Promoting ◽  
Stimulation Of ◽  
Cell Growth And Survival

G17 has growth promoting and antiapoptotic effects on the AR4–2J pancreatic acinar cell line. We previously reported that whereas MAPK regulates G17-stimulation of AR4–2J cell proliferation, Akt mediates the antiapoptotic action of G17. We examined the signal-transduction pathways mediating G17 stimulation of AR4–2J cell growth and survival. G17 activated the small GTP binding proteins Ras, Rac, Rho, and Cdc42. Transduction of the cells with adenoviral vectors expressing dominant negative Akt, Ras, Rho, and Cdc42 but not dominant negative Rac inhibited AR4–2J cell proliferation and survival. Both exoenzyme C3 from Clostridium botulinum (C3), a toxin known to inactivate Rho, and PD98059, a MAPK inhibitor, reversed G17 inhibition of AR4–2J cell apoptosis. G17 induction of Akt activation was reduced by >60% by both dominant negative Ras and Rho and by 30% by dominant negative Cdc42. In contrast, G17-stimulated MAPK activation was blocked by >80% by dominant negative Ras but not by dominant negative Rho and Cdc42. Similar results were observed in the presence of C3. Dominant negative Rac failed to affect G17 induction of both Akt and MAPK, whereas it inhibited sorbitol by almost 50% but not G17-stimulated activation of p38 kinase. Thus G17 promotes AR4–2J cell growth and survival through the activation of multiple GTP binding proteins, which, in turn, regulate different protein kinase cascades. Whereas Ras activates Akt and MAPK, Rho and Cdc42 appear to regulate Akt and possibly other as yet unidentified kinases mediating the growth-stimulatory actions of G17.

scholarly journals Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins.

1990 ◽  
Vol 111 (6) ◽  
pp. 2893-2908 ◽  
Author(s):  
D Gravotta ◽  
M Adesnik ◽  
D D Sabatini
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Binding Proteins ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Surface ◽  
Gtp Binding Proteins ◽  
Trans Golgi Network ◽  
Gtp Binding ◽  
Golgi Network

A procedure employing streptolysin O to effect the selective permeabilization of either the apical or basolateral plasma membrane domains of MDCK cell monolayers grown on a filter support was developed which permeabilizes the entire monolayer, leaves the opposite cell surface domain intact, and does not abolish the integrity of the tight junctions. This procedure renders the cell interior accessible to exogenous macromolecules and impermeant reagents, permitting the examination of their effects on membrane protein transport to the intact surface. The last stages of the transport of the influenza virus hemagglutinin (HA) to the apical surface were studied in pulse-labeled, virus-infected MDCK cells that were incubated at 19.5 degrees C for 90 min to accumulate newly synthesized HA in the trans-Golgi network (TGN), before raising the temperature to 35 degrees C to allow synchronized transport to the plasma membrane. In cells permeabilized immediately after the cold block, 50% of the intracellular HA molecules were subsequently delivered to the apical surface. This transport was dependent on the presence of an exogenous ATP supply and was markedly inhibited by the addition of GTP-gamma-S at the time of permeabilization. On the other hand, the GTP analogue had no effect when it was added to cells that, after the cold block, were incubated for 15 min at 35 degrees C before permeabilization, even though at this time most HA molecules were still intracellular and their appearance at the cell surface was largely dependent on exogenous ATP. These findings indicate that GTP-binding proteins are involved in the constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged early in this process. Transport of HA to the cell surface could be made dependent on the addition of exogenous cytosol when, after permeabilization, cells were washed to remove endogenous cytosolic components. This opens the way towards the identification of cell components that mediate the sorting of apical and basolateral membrane components in the TGN and their polarized delivery to the cell surface.

scholarly journals Atypical Protein Kinases Cλ and -ζ Associate with the GTP-Binding Protein Cdc42 and Mediate Stress Fiber Loss

2000 ◽  
Vol 20 (8) ◽  
pp. 2880-2889 ◽  
Author(s):  
Matthew P. Coghlan ◽  
Margaret M. Chou ◽  
Christopher L. Carpenter
Keyword(s):  
Protein Kinases ◽  
Binding Proteins ◽  
Dominant Negative ◽  
Stress Fibers ◽  
Regulation Of Transcription ◽  
Dependent Manner ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Intracellular Signals ◽  
Rho Family

ABSTRACT Both the Rho family of low-molecular-weight GTP-binding proteins and protein kinases C (PKCs) mediate responses to a variety of extracellular and intracellular signals. They share many downstream targets, including remodeling of the actin cytoskeleton, activation of p70S6 kinase and c-jun N-terminal kinase (JNK), and regulation of transcription and cell proliferation. We therefore investigated whether Rho family GTP-binding proteins bind to PKCs. We found that Cdc42 associates with atypical PKCs (aPKCs) PKCζ and -λ in a GTP-dependent manner. The regulatory domain of the aPKCs mediates the interaction. Expression of activated Cdc42 results in the translocation of PKCλ from the nucleus into the cytosol, and Cdc42 and PKCλ colocalize at the plasma membrane and in the cytoplasm. Expression of activated Cdc42 leads to a loss of stress fibers, as does overexpression of either the wild type or an activated form of PKCλ. Kinase-dead PKCλ and -ζ constructs acted as dominant negatives and restored stress fibers in cells expressing the activated V12 Cdc42 mutant, indicating that Cdc42-dependent loss of stress fibers requires aPKCs. Kinase-dead PKCλ and -ζ and dominant-negative N17 Cdc42 also blocked Ras-induced loss of stress fibers, suggesting that this pathway may also be important for Ras-dependent cytoskeletal changes. N17 Rac did not block Ras-induced loss of stress fibers, nor did kinase-dead PKCλ block V12 Rac-stimulated loss of stress fibers. These results indicate that Cdc42 and Rac use different pathways to regulate stress fibers.

Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

1999 ◽  
Vol 82 (09) ◽  
pp. 1177-1181 ◽  
Author(s):  
Hubert de Leeuw ◽  
Pauline Wijers-Koster ◽  
Jan van Mourik ◽  
Jan Voorberg
Keyword(s):  
Endothelial Cells ◽  
Binding Proteins ◽  
Binding Protein ◽  
Control Release ◽  
Small Gtpase ◽  
Gtp Binding Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Gtp Binding Proteins ◽  
Dense Granules ◽  
Gtp Binding

SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

1998 ◽  
Vol 79 (04) ◽  
pp. 832-836 ◽  
Author(s):  
Thomas Fischer ◽  
Christina Duffy ◽  
Gilbert White
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Binding Proteins ◽  
Binding Protein ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

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