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LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to drive collagen remodeling during invasion.

Konen J, Wilkinson S, Lee B, Fu H, Zhou W, Jiang Y, Marcus AI - Mol. Biol. Cell (2016)

Bottom Line: The majority of LKB1 mutations are truncations that disrupt its kinase activity and remove its C-terminal domain (CTD).Instead, cell polarity is overseen by the kinase-independent function of its CTD and more specifically its farnesylation.This occurs through a mesenchymal-amoeboid morphological switch that signals through the Rho-GTPase RhoA.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, GA 30322 Graduate Program in Cancer Biology, Emory University, Atlanta, GA 30322.

No MeSH data available.


Related in: MedlinePlus

LKB1 differentially regulates RhoA and cdc42. (A) Representative Western blot showing RhoA activity assay over time in pLKO.1 and shLKB1 H1299 cells embedded in a collagen type I matrix. (B) Densitometry of Western blot from A normalized to total RhoA levels. (C) Representative Western blot of a cdc42 activity assay over time in pLKO.1 and shLKB1 H1299 cells embedded in a collagen type I matrix. (D) Densitometry of Western blot from C normalized to total cdc42 levels. (E) Representative Western blot showing RhoA activity assay in two dimensions in pLKO.1 and shLKB1 H1299 cells. (F) Densitometry of Western blot from E normalized to total RhoA levels. (G) Representative Western blot showing cdc42 activity assay in two dimensions in pLKO.1 and shLKB1 H1299 cells. (H) Densitometry of Western blot from G normalized to total cdc42 levels. (I) Representative RhoA activity assay of H157 cells stably expressing either empty GFP control or GFP-tagged, wild-type LKB1, LKB1 C430S, or the LKB1 C-terminal domain. (J) Densitometry of Western blot from I normalized to total RhoA levels. (K) Representative Western blot showing a cdc42 activity assay of H157 cells stably expressing either empty GFP control or GFP-tagged, wild-type LKB1, LKB1 C430S, or the LKB1 C-terminal domain. (L) Densitometry of Western blot from K normalized to total cdc42. Three experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.
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Figure 3: LKB1 differentially regulates RhoA and cdc42. (A) Representative Western blot showing RhoA activity assay over time in pLKO.1 and shLKB1 H1299 cells embedded in a collagen type I matrix. (B) Densitometry of Western blot from A normalized to total RhoA levels. (C) Representative Western blot of a cdc42 activity assay over time in pLKO.1 and shLKB1 H1299 cells embedded in a collagen type I matrix. (D) Densitometry of Western blot from C normalized to total cdc42 levels. (E) Representative Western blot showing RhoA activity assay in two dimensions in pLKO.1 and shLKB1 H1299 cells. (F) Densitometry of Western blot from E normalized to total RhoA levels. (G) Representative Western blot showing cdc42 activity assay in two dimensions in pLKO.1 and shLKB1 H1299 cells. (H) Densitometry of Western blot from G normalized to total cdc42 levels. (I) Representative RhoA activity assay of H157 cells stably expressing either empty GFP control or GFP-tagged, wild-type LKB1, LKB1 C430S, or the LKB1 C-terminal domain. (J) Densitometry of Western blot from I normalized to total RhoA levels. (K) Representative Western blot showing a cdc42 activity assay of H157 cells stably expressing either empty GFP control or GFP-tagged, wild-type LKB1, LKB1 C430S, or the LKB1 C-terminal domain. (L) Densitometry of Western blot from K normalized to total cdc42. Three experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Mentions: We next sought to understand the mechanism by which LKB1 regulates this amoeboid–mesenchymal invasion switch. Because amoeboid invasive motility is driven through a balance of RhoA and cdc42 activity (Huang et al., 1997; Parri and Chiarugi, 2010; Morley et al., 2014), we probed the activity of these GTPases in 3D spheroids. A time course of H1299 (wild-type LKB1) pLKO.1 control cells shows robust activation of both RhoA and cdc42 at the 5-h time point (Figure 3, A–D); however, the isogenic shLKB1 cells have reduced active cdc42 and RhoA. To enrich for motile cells, GTPase activation assays were performed in two dimensions. The data show that H1299 cells activate both RhoA and cdc42, but this activation is severely attenuated upon LKB1 loss (Figure 3, E–H). These data are consistent with previous reports showing that LKB1 depletion reduces cdc42 and RhoA activity in motile cells (Zhang et al., 2008; Xu et al., 2010, 2013) and suggest that LKB1-depleted cells do not rely on canonical Rho-GTPase activity.


LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to drive collagen remodeling during invasion.

Konen J, Wilkinson S, Lee B, Fu H, Zhou W, Jiang Y, Marcus AI - Mol. Biol. Cell (2016)

LKB1 differentially regulates RhoA and cdc42. (A) Representative Western blot showing RhoA activity assay over time in pLKO.1 and shLKB1 H1299 cells embedded in a collagen type I matrix. (B) Densitometry of Western blot from A normalized to total RhoA levels. (C) Representative Western blot of a cdc42 activity assay over time in pLKO.1 and shLKB1 H1299 cells embedded in a collagen type I matrix. (D) Densitometry of Western blot from C normalized to total cdc42 levels. (E) Representative Western blot showing RhoA activity assay in two dimensions in pLKO.1 and shLKB1 H1299 cells. (F) Densitometry of Western blot from E normalized to total RhoA levels. (G) Representative Western blot showing cdc42 activity assay in two dimensions in pLKO.1 and shLKB1 H1299 cells. (H) Densitometry of Western blot from G normalized to total cdc42 levels. (I) Representative RhoA activity assay of H157 cells stably expressing either empty GFP control or GFP-tagged, wild-type LKB1, LKB1 C430S, or the LKB1 C-terminal domain. (J) Densitometry of Western blot from I normalized to total RhoA levels. (K) Representative Western blot showing a cdc42 activity assay of H157 cells stably expressing either empty GFP control or GFP-tagged, wild-type LKB1, LKB1 C430S, or the LKB1 C-terminal domain. (L) Densitometry of Western blot from K normalized to total cdc42. Three experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.
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Figure 3: LKB1 differentially regulates RhoA and cdc42. (A) Representative Western blot showing RhoA activity assay over time in pLKO.1 and shLKB1 H1299 cells embedded in a collagen type I matrix. (B) Densitometry of Western blot from A normalized to total RhoA levels. (C) Representative Western blot of a cdc42 activity assay over time in pLKO.1 and shLKB1 H1299 cells embedded in a collagen type I matrix. (D) Densitometry of Western blot from C normalized to total cdc42 levels. (E) Representative Western blot showing RhoA activity assay in two dimensions in pLKO.1 and shLKB1 H1299 cells. (F) Densitometry of Western blot from E normalized to total RhoA levels. (G) Representative Western blot showing cdc42 activity assay in two dimensions in pLKO.1 and shLKB1 H1299 cells. (H) Densitometry of Western blot from G normalized to total cdc42 levels. (I) Representative RhoA activity assay of H157 cells stably expressing either empty GFP control or GFP-tagged, wild-type LKB1, LKB1 C430S, or the LKB1 C-terminal domain. (J) Densitometry of Western blot from I normalized to total RhoA levels. (K) Representative Western blot showing a cdc42 activity assay of H157 cells stably expressing either empty GFP control or GFP-tagged, wild-type LKB1, LKB1 C430S, or the LKB1 C-terminal domain. (L) Densitometry of Western blot from K normalized to total cdc42. Three experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.
Mentions: We next sought to understand the mechanism by which LKB1 regulates this amoeboid–mesenchymal invasion switch. Because amoeboid invasive motility is driven through a balance of RhoA and cdc42 activity (Huang et al., 1997; Parri and Chiarugi, 2010; Morley et al., 2014), we probed the activity of these GTPases in 3D spheroids. A time course of H1299 (wild-type LKB1) pLKO.1 control cells shows robust activation of both RhoA and cdc42 at the 5-h time point (Figure 3, A–D); however, the isogenic shLKB1 cells have reduced active cdc42 and RhoA. To enrich for motile cells, GTPase activation assays were performed in two dimensions. The data show that H1299 cells activate both RhoA and cdc42, but this activation is severely attenuated upon LKB1 loss (Figure 3, E–H). These data are consistent with previous reports showing that LKB1 depletion reduces cdc42 and RhoA activity in motile cells (Zhang et al., 2008; Xu et al., 2010, 2013) and suggest that LKB1-depleted cells do not rely on canonical Rho-GTPase activity.

Bottom Line: The majority of LKB1 mutations are truncations that disrupt its kinase activity and remove its C-terminal domain (CTD).Instead, cell polarity is overseen by the kinase-independent function of its CTD and more specifically its farnesylation.This occurs through a mesenchymal-amoeboid morphological switch that signals through the Rho-GTPase RhoA.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, GA 30322 Graduate Program in Cancer Biology, Emory University, Atlanta, GA 30322.

No MeSH data available.


Related in: MedlinePlus