km23-1/DYNLRB1 regulation of MEK/ERK signaling and R-Ras in invasive human colorectal cancer cells

Asif Raza, Madhu S. Pandey, Qunyan Jin, Kathleen Mulder

Research output: Contribution to journalArticle

Abstract

We previously found that km23-1/DYNLRB1 is required for transforming growth factor-β (TGFβ) production through Ras/ERK pathways in TGFβ-sensitive epithelial cells and in human colorectal cancer (CRC) cells. Here we demonstrate that km23-1/DYNLRB1 is required for mitogen-activated protein kinase kinase (MEK) activation in human CRC cells, detected by km23-1/DYNLRB1-siRNA inhibition of phospho-(p)-MEK immunostaining in RKO cells. Furthermore, we show that CRISPR-Cas9 knock-out (KO) of km23-1/DYNLRB1 reduced cell migration in two additional CRC models, HCT116 and DLD-1. Of interest, in contrast to our previous work showing that dynein motor activity was required for TGFβ-mediated nuclear translocation of Smad2, in the current report, we demonstrate for the first time that disruption of dynein motor activity did not reduce TGFβ-mediated activation of MEK1/2 or c-Jun N-terminal kinase (JNK). Moreover, size exclusion chromatography of RKO cell lysates revealed that B-Raf, extracellular signal-regulated kinase (ERK), and p-ERK were not present in the large molecular weight fractions containing dynein holocomplex components. Furthermore, sucrose gradient fractionation of cell lysates from both HCT116 and CBS CRC cells demonstrated that km23-1/DYNLRB1 co-sedimented with Ras, p-ERK, and ERK in fractions that did not contain components of holo-dynein. Thus, km23-1/DYNLRB1 may be associated with activated Ras/ERK signaling complexes in cell compartments that do not contain the dynein holoprotein complex, suggesting dynein-independent km23-1/DYNLRB1 functions in Ras/ERK signaling. Finally, of the Ras isoforms, R-Ras is most often associated with cell migration, adhesion, and protrusive activity. Here, we show that a significant fraction of km23-1/DYNLRB1 and RRas wase co-localized at the protruding edges of migrating HCT116 cells, suggesting an important role for the km23-1/DYNLRB1-R-Ras complex in CRC invasion.

Original languageEnglish (US)
JournalCell Biology International
DOIs
StateAccepted/In press - Jan 1 2019

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Mitogen-Activated Protein Kinase Kinases
Extracellular Signal-Regulated MAP Kinases
Dyneins
Colorectal Neoplasms
Transforming Growth Factors
Cell Movement
Motor Activity
Mitogen-Activated Protein Kinase 9
Clustered Regularly Interspaced Short Palindromic Repeats
HCT116 Cells
Cell Fractionation
Cell Adhesion
Small Interfering RNA
Gel Chromatography
Sucrose
Protein Isoforms
Molecular Weight
Epithelial Cells

All Science Journal Classification (ASJC) codes

  • Cell Biology

Cite this

@article{96c841b8ba9640069fa3c94fbf4d2eb9,
title = "km23-1/DYNLRB1 regulation of MEK/ERK signaling and R-Ras in invasive human colorectal cancer cells",
abstract = "We previously found that km23-1/DYNLRB1 is required for transforming growth factor-β (TGFβ) production through Ras/ERK pathways in TGFβ-sensitive epithelial cells and in human colorectal cancer (CRC) cells. Here we demonstrate that km23-1/DYNLRB1 is required for mitogen-activated protein kinase kinase (MEK) activation in human CRC cells, detected by km23-1/DYNLRB1-siRNA inhibition of phospho-(p)-MEK immunostaining in RKO cells. Furthermore, we show that CRISPR-Cas9 knock-out (KO) of km23-1/DYNLRB1 reduced cell migration in two additional CRC models, HCT116 and DLD-1. Of interest, in contrast to our previous work showing that dynein motor activity was required for TGFβ-mediated nuclear translocation of Smad2, in the current report, we demonstrate for the first time that disruption of dynein motor activity did not reduce TGFβ-mediated activation of MEK1/2 or c-Jun N-terminal kinase (JNK). Moreover, size exclusion chromatography of RKO cell lysates revealed that B-Raf, extracellular signal-regulated kinase (ERK), and p-ERK were not present in the large molecular weight fractions containing dynein holocomplex components. Furthermore, sucrose gradient fractionation of cell lysates from both HCT116 and CBS CRC cells demonstrated that km23-1/DYNLRB1 co-sedimented with Ras, p-ERK, and ERK in fractions that did not contain components of holo-dynein. Thus, km23-1/DYNLRB1 may be associated with activated Ras/ERK signaling complexes in cell compartments that do not contain the dynein holoprotein complex, suggesting dynein-independent km23-1/DYNLRB1 functions in Ras/ERK signaling. Finally, of the Ras isoforms, R-Ras is most often associated with cell migration, adhesion, and protrusive activity. Here, we show that a significant fraction of km23-1/DYNLRB1 and RRas wase co-localized at the protruding edges of migrating HCT116 cells, suggesting an important role for the km23-1/DYNLRB1-R-Ras complex in CRC invasion.",
author = "Asif Raza and Pandey, {Madhu S.} and Qunyan Jin and Kathleen Mulder",
year = "2019",
month = "1",
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language = "English (US)",
journal = "Cell Biology International",
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km23-1/DYNLRB1 regulation of MEK/ERK signaling and R-Ras in invasive human colorectal cancer cells. / Raza, Asif; Pandey, Madhu S.; Jin, Qunyan; Mulder, Kathleen.

In: Cell Biology International, 01.01.2019.

Research output: Contribution to journalArticle

TY - JOUR

T1 - km23-1/DYNLRB1 regulation of MEK/ERK signaling and R-Ras in invasive human colorectal cancer cells

AU - Raza, Asif

AU - Pandey, Madhu S.

AU - Jin, Qunyan

AU - Mulder, Kathleen

PY - 2019/1/1

Y1 - 2019/1/1

N2 - We previously found that km23-1/DYNLRB1 is required for transforming growth factor-β (TGFβ) production through Ras/ERK pathways in TGFβ-sensitive epithelial cells and in human colorectal cancer (CRC) cells. Here we demonstrate that km23-1/DYNLRB1 is required for mitogen-activated protein kinase kinase (MEK) activation in human CRC cells, detected by km23-1/DYNLRB1-siRNA inhibition of phospho-(p)-MEK immunostaining in RKO cells. Furthermore, we show that CRISPR-Cas9 knock-out (KO) of km23-1/DYNLRB1 reduced cell migration in two additional CRC models, HCT116 and DLD-1. Of interest, in contrast to our previous work showing that dynein motor activity was required for TGFβ-mediated nuclear translocation of Smad2, in the current report, we demonstrate for the first time that disruption of dynein motor activity did not reduce TGFβ-mediated activation of MEK1/2 or c-Jun N-terminal kinase (JNK). Moreover, size exclusion chromatography of RKO cell lysates revealed that B-Raf, extracellular signal-regulated kinase (ERK), and p-ERK were not present in the large molecular weight fractions containing dynein holocomplex components. Furthermore, sucrose gradient fractionation of cell lysates from both HCT116 and CBS CRC cells demonstrated that km23-1/DYNLRB1 co-sedimented with Ras, p-ERK, and ERK in fractions that did not contain components of holo-dynein. Thus, km23-1/DYNLRB1 may be associated with activated Ras/ERK signaling complexes in cell compartments that do not contain the dynein holoprotein complex, suggesting dynein-independent km23-1/DYNLRB1 functions in Ras/ERK signaling. Finally, of the Ras isoforms, R-Ras is most often associated with cell migration, adhesion, and protrusive activity. Here, we show that a significant fraction of km23-1/DYNLRB1 and RRas wase co-localized at the protruding edges of migrating HCT116 cells, suggesting an important role for the km23-1/DYNLRB1-R-Ras complex in CRC invasion.

AB - We previously found that km23-1/DYNLRB1 is required for transforming growth factor-β (TGFβ) production through Ras/ERK pathways in TGFβ-sensitive epithelial cells and in human colorectal cancer (CRC) cells. Here we demonstrate that km23-1/DYNLRB1 is required for mitogen-activated protein kinase kinase (MEK) activation in human CRC cells, detected by km23-1/DYNLRB1-siRNA inhibition of phospho-(p)-MEK immunostaining in RKO cells. Furthermore, we show that CRISPR-Cas9 knock-out (KO) of km23-1/DYNLRB1 reduced cell migration in two additional CRC models, HCT116 and DLD-1. Of interest, in contrast to our previous work showing that dynein motor activity was required for TGFβ-mediated nuclear translocation of Smad2, in the current report, we demonstrate for the first time that disruption of dynein motor activity did not reduce TGFβ-mediated activation of MEK1/2 or c-Jun N-terminal kinase (JNK). Moreover, size exclusion chromatography of RKO cell lysates revealed that B-Raf, extracellular signal-regulated kinase (ERK), and p-ERK were not present in the large molecular weight fractions containing dynein holocomplex components. Furthermore, sucrose gradient fractionation of cell lysates from both HCT116 and CBS CRC cells demonstrated that km23-1/DYNLRB1 co-sedimented with Ras, p-ERK, and ERK in fractions that did not contain components of holo-dynein. Thus, km23-1/DYNLRB1 may be associated with activated Ras/ERK signaling complexes in cell compartments that do not contain the dynein holoprotein complex, suggesting dynein-independent km23-1/DYNLRB1 functions in Ras/ERK signaling. Finally, of the Ras isoforms, R-Ras is most often associated with cell migration, adhesion, and protrusive activity. Here, we show that a significant fraction of km23-1/DYNLRB1 and RRas wase co-localized at the protruding edges of migrating HCT116 cells, suggesting an important role for the km23-1/DYNLRB1-R-Ras complex in CRC invasion.

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