scholarly journals Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis

2003 ◽  
Vol 160 (3) ◽  
pp. 375-385 ◽  
Author(s):  
Supriya Srinivasan ◽  
Fei Wang ◽  
Suzana Glavas ◽  
Alexander Ott ◽  
Fred Hofmann ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Positive Feedback ◽  
Feedback Loop ◽  
Dominant Role ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Dominant Negative ◽  
Ph Domain ◽  
Positive Feedback Loop ◽  
Constitutively Active

Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)P3 (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42.

PGE2 potentiates tonicity-induced COX-2 expression in renal medullary cells in a positive feedback loop involving EP2-cAMP-PKA signaling

2009 ◽  
Vol 296 (1) ◽  
pp. C75-C87 ◽  
Author(s):  
Daniela Steinert ◽  
Christoph Küper ◽  
Helmut Bartels ◽  
Franz-X. Beck ◽  
Wolfgang Neuhofer
Keyword(s):  
Osmotic Stress ◽  
Cell Survival ◽  
Positive Feedback ◽  
Feedback Loop ◽  
Mdck Cells ◽  
Dominant Negative ◽  
Dibutyryl Camp ◽  
Positive Feedback Loop ◽  
Cox 2 ◽  
Medullary Cells

Cyooxygenase-2 (COX-2)-derived PGE2 is critical for the integrity and function of renal medullary cells during antidiuresis. The present study extended our previous finding that tonicity-induced COX-2 expression is further stimulated by the major COX-2 product PGE2 and investigated the underlying signaling pathways and the functional relevance of this phenomenon. Hyperosmolality stimulated COX-2 expression and activity in Madin-Darby canine kidney (MDCK) cells, a response that was further increased by PGE2-cAMP signaling, suggesting the existence of a positive feedback loop. This effect was diminished by AH-6809, an EP2 antagonist, and by the PKA inhibitor H-89, but not by AH-23848, an EP4 antagonist. The effect of PGE2 was mimicked by forskolin and dibutyryl-cAMP, suggesting that the stimulatory effect of PGE2 on COX-2 is mediated by a cAMP-PKA-dependent mechanism. Accordingly, cAMP-responsive element (CRE)-driven reporter activity paralleled the effects of PGE2, AH-6809, AH-23848, H-89, forskolin, and dibutyryl-cAMP on COX-2 expression. In addition, the stimulatory effect of PGE2 on tonicity-induced COX-2 expression was blunted in cells transfected with dominant-negative CRE binding (CREB) protein, as was the case in a COX-2 promoter reporter construct in which a putative CRE was deleted. Furthermore, PGE2 resulted in PKA-dependent phosphorylation of the pro-apoptotic protein Bad at Ser155, a mechanism that is known to inactivate Bad, which coincided with reduced caspase-3 activity during osmotic stress. Conversely, pharmacological interruption of the PGE2-EP2-cAMP-PKA pathway abolished Ser155 phosphorylation of Bad and blunted the protective effect of PGE2 on cell survival during osmotic stress. These observations indicate the existence of a positive feedback loop of PGE2 on COX-2 expression during osmotic stress, an effect that apparently is mediated by EP2-cAMP-PKA signaling, and that contributes to cell survival under hypertonic conditions.

scholarly journals Paxillin phosphorylation at Ser273 localizes a GIT1–PIX–PAK complex and regulates adhesion and protrusion dynamics

2006 ◽  
Vol 173 (4) ◽  
pp. 587-589 ◽  
Author(s):  
Anjana Nayal ◽  
Donna J. Webb ◽  
Claire M. Brown ◽  
Erik M. Schaefer ◽  
Miguel Vicente-Manzanares ◽  
...  
Keyword(s):  
Positive Feedback ◽  
Feedback Loop ◽  
Myosin Ii ◽  
Adhesion Formation ◽  
Leading Edge ◽  
Positive Feedback Loop ◽  
Motile Cells ◽  
Downstream Effectors ◽  
P21 Activated Kinase ◽  
Migrating Cells

Continuous adhesion formation and disassembly (adhesion turnover) in the protrusions of migrating cells is regulated by unclear mechanisms. We show that p21-activated kinase (PAK)–induced phosphorylation of serine 273 in paxillin is a critical regulator of this turnover. Paxillin-S273 phosphorylation dramatically increases migration, protrusion, and adhesion turnover by increasing paxillin–GIT1 binding and promoting the localization of a GIT1–PIX–PAK signaling module near the leading edge. Mutants that interfere with the formation of this ternary module abrogate the effects of paxillin-S273 phosphorylation. PAK-dependent paxillin-S273 phosphorylation functions in a positive-feedback loop, as active PAK, active Rac, and myosin II activity are all downstream effectors of this turnover pathway. Finally, our studies led us to identify in highly motile cells a class of small adhesions that reside near the leading edge, turnover in 20–30 s, and resemble those seen with paxillin-S273 phosphorylation. These adhesions appear to be regulated by the GIT1–PIX–PAK module near the leading edge.

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