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

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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Ca2+ influx is an essential component of the positive-feedback loop that maintains leading-edge structure and activity in macrophages

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0707719104 ◽

2007 ◽

Vol 104 (41) ◽

pp. 16176-16181 ◽

Cited By ~ 95

Author(s):

J. H. Evans ◽

J. J. Falke

Keyword(s):

Positive Feedback ◽

Feedback Loop ◽

Leading Edge ◽

Positive Feedback Loop ◽

Edge Structure ◽

Essential Component ◽

Structure And Activity

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

The Journal of Cell Biology ◽

10.1083/jcb.200208179 ◽

2003 ◽

Vol 160 (3) ◽

pp. 375-385 ◽

Cited By ~ 305

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.

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A SMYD3/ITGB6/TGFβ1 Positive Feedback Loop Promotes the Invasion and Adhesion of Ovarian Cancer Spheroids

Frontiers in Oncology ◽

10.3389/fonc.2021.690618 ◽

2021 ◽

Vol 11 ◽

Author(s):

Yahui Jiang ◽

Tianyu Zhou ◽

Yiwen Shi ◽

Weiwei Feng ◽

Tianjiao Lyu

Keyword(s):

Ovarian Cancer ◽

Positive Feedback ◽

Feedback Loop ◽

Cancer Metastasis ◽

Luciferase Reporter ◽

Positive Feedback Loop ◽

Downstream Effectors ◽

Reporter Assays ◽

Cancer Spheroids ◽

E Cadherin

BackgroundImplantation metastasis is the main means of dissemination in ovarian cancer. Our previous studies showed that SET and MYND domain-containing protein 3 (SMYD3) expression was higher in ovarian cancer spheroids than in monolayers. SMYD3 enhancement of spheroid invasion and adhesion is mediated by the downstream effectors ITGB6 and ITGAM. However, the potential mechanisms underlying the SMYD3/integrin-mediated invasion and adhesion of spheroids still need to be explored.MethodsWestern blotting was used to examine the expression of SMYD3, ITGB6 and downstream molecules under different treatments. Immunofluorescence was used to detect the expression of F-actin, E-cadherin and N-cadherin. Anti-ITGB6 antibody-based inhibition and dual-luciferase reporter assays were used to confirm the binding between ITGB6 and latent TGFβ1. Transwell invasion, adherence and 3D tumor spheroid invasion assays were employed to test the effects of TGFβ1 on the invasion and adhesion of ovarian cancer spheroids. ELISA was performed to assess the release of latent TGFβ1 from ovarian cancer spheroids.ResultsSMYD3 and ITGB6 activated the TGFβ1/Smad3 pathway and then induced the upregulation of Snail, Vimentin and N-cadherin and the downregulation of E-cadherin in 3D-cultured ovarian cancer spheroids. In this process, latent TGFβ1 could bind to ITGB6 and become activated to stimulate the Smad3 pathway. Moreover, SMYD3 and ITGB6 could facilitate the release of latent TGFβ1 from 3D-cultured ovarian cancer spheroids. Interestingly, TGFβ1 could promote the expression of SMYD3 and ITGB6 via feedback. This positive feedback loop could further amplify the biological effect and promote the invasion and adhesion of ovarian cancer spheroids.ConclusionOur results demonstrated that the SMYD3/ITGB6/TGFβ1-Smad3 positive feedback loop could promote the invasion and adhesion of ovarian cancer spheroids by upregulating the expression of N-cadherin, Snail, and Vimentin and downregulating the expression of E-cadherin. Thus, our study unmasked the mechanism of SMYD3- and ITGB6-induced ovarian cancer metastasis and provides new ideas for targeted ovarian cancer treatment.

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