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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 regulates pFAK activity through its kinase domain. (A) Spheroids of H157 cells stably expressing either empty GFP control or GFP-tagged LKB1 constructs embedded in a collagen type I matrix. After 24 h, cells were fixed and stained by immunofluorescence for pFAKY397, GFP, and DAPI. (B) Total number of pFAKY397 sites for each experimental group in A was quantified. (C) Expression of pFAKY397 was examined by immunofluorescence of spheroids after 24 h of invasion. DAPI was used to stain the nuclei of the cells. (D) The total number of pFAKY397 sites per cell was quantified from the images obtained in C. ****p ≤ 0.0001. (E) Western blot showing pFAKY397 expression after MARK1 siRNA depletion in H1792, H1299, and H157 LKB1 WT cells compared with scrambled control siRNA. GAPDH was used as a loading control. Bottom, densitometry analysis of phospho to total FAK ratio (left) and relative MARK1 expression (right) in control siRNA– and MARK1 siRNA–treated cells.
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Figure 5: LKB1 regulates pFAK activity through its kinase domain. (A) Spheroids of H157 cells stably expressing either empty GFP control or GFP-tagged LKB1 constructs embedded in a collagen type I matrix. After 24 h, cells were fixed and stained by immunofluorescence for pFAKY397, GFP, and DAPI. (B) Total number of pFAKY397 sites for each experimental group in A was quantified. (C) Expression of pFAKY397 was examined by immunofluorescence of spheroids after 24 h of invasion. DAPI was used to stain the nuclei of the cells. (D) The total number of pFAKY397 sites per cell was quantified from the images obtained in C. ****p ≤ 0.0001. (E) Western blot showing pFAKY397 expression after MARK1 siRNA depletion in H1792, H1299, and H157 LKB1 WT cells compared with scrambled control siRNA. GAPDH was used as a loading control. Bottom, densitometry analysis of phospho to total FAK ratio (left) and relative MARK1 expression (right) in control siRNA– and MARK1 siRNA–treated cells.

Mentions: The present data show that the LKB1 CTD is responsible for controlling the amoeboid–mesenchymal switch during 3D invasion. We and others have also shown that LKB1 loss results in an increase in adhesion signaling, most notably through hyperactive FAK signaling (Ji et al., 2007; Carretero et al., 2010; Kline et al., 2013; Goodwin et al., 2014). Thus we sought to determine whether this amoeboid–mesenchymal switch is related to adhesion signaling by using our panel of H157 stable cells (Figure 2B) to assess pFAK397 activity. We found, using immunofluorescence, that compared to empty GFP control cells, H157 cells expressing wild-type LKB1 showed repression of the total number of pFAK397 sites (Figure 5, A and B). This result is consistent with previous findings that LKB1 is a pFAK repressor in lung cancer cells (Kline et al., 2013; Goodwin et al., 2014). Of interest, this repression was not dependent on LKB1 farnesylation, as mutation of the LKB1 C430 farnesylation motif had no effect on the ability of LKB1 to repress pFAK397 (Figure 5, A and B). In contrast, on reexpression of either the K78I kinase-dead mutant or the K78I-C430S double mutant LKB1, pFAK397 expression was not repressed and remained at levels similar to those for empty GFP control cells (Figure 5, A and B). Whereas LKB1 served to repress the total number of pFAK397 sites per cell, the mean intensity of each pFAK site was similar across all cell lines (Supplemental Figure S5). These data suggest that the kinase activity of LKB1, but not farnesylation, is necessary for LKB1 to repress FAK during 3D invasion.


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 regulates pFAK activity through its kinase domain. (A) Spheroids of H157 cells stably expressing either empty GFP control or GFP-tagged LKB1 constructs embedded in a collagen type I matrix. After 24 h, cells were fixed and stained by immunofluorescence for pFAKY397, GFP, and DAPI. (B) Total number of pFAKY397 sites for each experimental group in A was quantified. (C) Expression of pFAKY397 was examined by immunofluorescence of spheroids after 24 h of invasion. DAPI was used to stain the nuclei of the cells. (D) The total number of pFAKY397 sites per cell was quantified from the images obtained in C. ****p ≤ 0.0001. (E) Western blot showing pFAKY397 expression after MARK1 siRNA depletion in H1792, H1299, and H157 LKB1 WT cells compared with scrambled control siRNA. GAPDH was used as a loading control. Bottom, densitometry analysis of phospho to total FAK ratio (left) and relative MARK1 expression (right) in control siRNA– and MARK1 siRNA–treated cells.
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Figure 5: LKB1 regulates pFAK activity through its kinase domain. (A) Spheroids of H157 cells stably expressing either empty GFP control or GFP-tagged LKB1 constructs embedded in a collagen type I matrix. After 24 h, cells were fixed and stained by immunofluorescence for pFAKY397, GFP, and DAPI. (B) Total number of pFAKY397 sites for each experimental group in A was quantified. (C) Expression of pFAKY397 was examined by immunofluorescence of spheroids after 24 h of invasion. DAPI was used to stain the nuclei of the cells. (D) The total number of pFAKY397 sites per cell was quantified from the images obtained in C. ****p ≤ 0.0001. (E) Western blot showing pFAKY397 expression after MARK1 siRNA depletion in H1792, H1299, and H157 LKB1 WT cells compared with scrambled control siRNA. GAPDH was used as a loading control. Bottom, densitometry analysis of phospho to total FAK ratio (left) and relative MARK1 expression (right) in control siRNA– and MARK1 siRNA–treated cells.
Mentions: The present data show that the LKB1 CTD is responsible for controlling the amoeboid–mesenchymal switch during 3D invasion. We and others have also shown that LKB1 loss results in an increase in adhesion signaling, most notably through hyperactive FAK signaling (Ji et al., 2007; Carretero et al., 2010; Kline et al., 2013; Goodwin et al., 2014). Thus we sought to determine whether this amoeboid–mesenchymal switch is related to adhesion signaling by using our panel of H157 stable cells (Figure 2B) to assess pFAK397 activity. We found, using immunofluorescence, that compared to empty GFP control cells, H157 cells expressing wild-type LKB1 showed repression of the total number of pFAK397 sites (Figure 5, A and B). This result is consistent with previous findings that LKB1 is a pFAK repressor in lung cancer cells (Kline et al., 2013; Goodwin et al., 2014). Of interest, this repression was not dependent on LKB1 farnesylation, as mutation of the LKB1 C430 farnesylation motif had no effect on the ability of LKB1 to repress pFAK397 (Figure 5, A and B). In contrast, on reexpression of either the K78I kinase-dead mutant or the K78I-C430S double mutant LKB1, pFAK397 expression was not repressed and remained at levels similar to those for empty GFP control cells (Figure 5, A and B). Whereas LKB1 served to repress the total number of pFAK397 sites per cell, the mean intensity of each pFAK site was similar across all cell lines (Supplemental Figure S5). These data suggest that the kinase activity of LKB1, but not farnesylation, is necessary for LKB1 to repress FAK during 3D invasion.

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