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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 cellular polarization through its C-terminal domain in a farnesylation-dependent manner. (A) LKB1 consists of a central kinase domain with a C-terminal farnesylation motif. Schematic of LKB1 mutations in lung adenocarcinoma patients; data adapted from cBioPortal (www.cbioportal.org). Red, truncating mutations; green, missense. (B) Schematic showing H157 (NSCLC, LKB1-) cells that were generated stably expressing GFP-tagged, wild-type LKB1, a C430S mutation to disrupt farnesylation, a K78I kinase-dead mutation, a double mutation with both K78I and C430S, the CTD alone, or the CTD alone with a C430S mutation. (C) Western blot probed with a GFP antibody verifying expression of the H157 stable cells. (D) Immuno­fluorescence of H157 spheroids embedded in collagen and stained with phalloidin. Amoeboid and mesenchymal morphologies (described in Figure 1) were quantified as a percentage back to the total number of cells invaded in each spheroid. Four spheroids. Scale, 20 μm. Arrows, mesenchymal cells; arrowheads, amoeboid cells. (E) The percentage of mesenchymal cells was quantified for each cell line at 24 h postembedding. (F) Each cell line was tracked over time. Cell tracks were plotted from a single point of origin. (G) Meandering index (defined as the linear distance divided by the total path length) was calculated using the cell tracks from F; 30 cells. ***p ≤ 0.001 and ****p ≤ 0.0001.
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Figure 2: LKB1 regulates cellular polarization through its C-terminal domain in a farnesylation-dependent manner. (A) LKB1 consists of a central kinase domain with a C-terminal farnesylation motif. Schematic of LKB1 mutations in lung adenocarcinoma patients; data adapted from cBioPortal (www.cbioportal.org). Red, truncating mutations; green, missense. (B) Schematic showing H157 (NSCLC, LKB1-) cells that were generated stably expressing GFP-tagged, wild-type LKB1, a C430S mutation to disrupt farnesylation, a K78I kinase-dead mutation, a double mutation with both K78I and C430S, the CTD alone, or the CTD alone with a C430S mutation. (C) Western blot probed with a GFP antibody verifying expression of the H157 stable cells. (D) Immuno­fluorescence of H157 spheroids embedded in collagen and stained with phalloidin. Amoeboid and mesenchymal morphologies (described in Figure 1) were quantified as a percentage back to the total number of cells invaded in each spheroid. Four spheroids. Scale, 20 μm. Arrows, mesenchymal cells; arrowheads, amoeboid cells. (E) The percentage of mesenchymal cells was quantified for each cell line at 24 h postembedding. (F) Each cell line was tracked over time. Cell tracks were plotted from a single point of origin. (G) Meandering index (defined as the linear distance divided by the total path length) was calculated using the cell tracks from F; 30 cells. ***p ≤ 0.001 and ****p ≤ 0.0001.

Mentions: Because the majority of LKB1 mutations in lung cancer patients are truncations (Cancer Genome Atlas Research Network, 2014; Figure 2A), we made a series of stable cells reexpressing GFP-tagged LKB1 mutants and domain truncates (Figure 2, B and C) to determine whether they could induce mesenchymal invasion in both H157 LKB1- human lung cancer cells and HeLa (LKB1- cervical cancer) cells. Based on the use of 3D invasion assays of spheroids embedded in collagen, a full-length, wild type LKB1 induced mesenchymal polarization during invasion as compared with empty GFP control (Figure 2, D and E, and Supplemental Figure S2), confirming the data seen with the transient transfections (Figure 1D). Similarly, H157 cells reexpressing an LKB1 K78I kinase-dead mutant (Supplemental Figure S3) also exhibited mesenchymal polarity, indicating that kinase activity is not required for promoting mesenchymal polarization. In contrast, a C430S farnesylation mutant or a K78I and C430S double mutant was unable to significantly restore mesenchymal polarization over empty GFP control, highlighting the role of LKB1 farnesylation in promoting mesenchymal polarization during invasion in a kinase-independent manner.


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 cellular polarization through its C-terminal domain in a farnesylation-dependent manner. (A) LKB1 consists of a central kinase domain with a C-terminal farnesylation motif. Schematic of LKB1 mutations in lung adenocarcinoma patients; data adapted from cBioPortal (www.cbioportal.org). Red, truncating mutations; green, missense. (B) Schematic showing H157 (NSCLC, LKB1-) cells that were generated stably expressing GFP-tagged, wild-type LKB1, a C430S mutation to disrupt farnesylation, a K78I kinase-dead mutation, a double mutation with both K78I and C430S, the CTD alone, or the CTD alone with a C430S mutation. (C) Western blot probed with a GFP antibody verifying expression of the H157 stable cells. (D) Immuno­fluorescence of H157 spheroids embedded in collagen and stained with phalloidin. Amoeboid and mesenchymal morphologies (described in Figure 1) were quantified as a percentage back to the total number of cells invaded in each spheroid. Four spheroids. Scale, 20 μm. Arrows, mesenchymal cells; arrowheads, amoeboid cells. (E) The percentage of mesenchymal cells was quantified for each cell line at 24 h postembedding. (F) Each cell line was tracked over time. Cell tracks were plotted from a single point of origin. (G) Meandering index (defined as the linear distance divided by the total path length) was calculated using the cell tracks from F; 30 cells. ***p ≤ 0.001 and ****p ≤ 0.0001.
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Related In: Results  -  Collection

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Figure 2: LKB1 regulates cellular polarization through its C-terminal domain in a farnesylation-dependent manner. (A) LKB1 consists of a central kinase domain with a C-terminal farnesylation motif. Schematic of LKB1 mutations in lung adenocarcinoma patients; data adapted from cBioPortal (www.cbioportal.org). Red, truncating mutations; green, missense. (B) Schematic showing H157 (NSCLC, LKB1-) cells that were generated stably expressing GFP-tagged, wild-type LKB1, a C430S mutation to disrupt farnesylation, a K78I kinase-dead mutation, a double mutation with both K78I and C430S, the CTD alone, or the CTD alone with a C430S mutation. (C) Western blot probed with a GFP antibody verifying expression of the H157 stable cells. (D) Immuno­fluorescence of H157 spheroids embedded in collagen and stained with phalloidin. Amoeboid and mesenchymal morphologies (described in Figure 1) were quantified as a percentage back to the total number of cells invaded in each spheroid. Four spheroids. Scale, 20 μm. Arrows, mesenchymal cells; arrowheads, amoeboid cells. (E) The percentage of mesenchymal cells was quantified for each cell line at 24 h postembedding. (F) Each cell line was tracked over time. Cell tracks were plotted from a single point of origin. (G) Meandering index (defined as the linear distance divided by the total path length) was calculated using the cell tracks from F; 30 cells. ***p ≤ 0.001 and ****p ≤ 0.0001.
Mentions: Because the majority of LKB1 mutations in lung cancer patients are truncations (Cancer Genome Atlas Research Network, 2014; Figure 2A), we made a series of stable cells reexpressing GFP-tagged LKB1 mutants and domain truncates (Figure 2, B and C) to determine whether they could induce mesenchymal invasion in both H157 LKB1- human lung cancer cells and HeLa (LKB1- cervical cancer) cells. Based on the use of 3D invasion assays of spheroids embedded in collagen, a full-length, wild type LKB1 induced mesenchymal polarization during invasion as compared with empty GFP control (Figure 2, D and E, and Supplemental Figure S2), confirming the data seen with the transient transfections (Figure 1D). Similarly, H157 cells reexpressing an LKB1 K78I kinase-dead mutant (Supplemental Figure S3) also exhibited mesenchymal polarity, indicating that kinase activity is not required for promoting mesenchymal polarization. In contrast, a C430S farnesylation mutant or a K78I and C430S double mutant was unable to significantly restore mesenchymal polarization over empty GFP control, highlighting the role of LKB1 farnesylation in promoting mesenchymal polarization during invasion in a kinase-independent manner.

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