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 M. Mulder

Research output: Contribution to journalArticlepeer-review

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)
Pages (from-to)155-165
Number of pages11
JournalCell Biology International
Volume44
Issue number1
DOIs
StatePublished - Jan 1 2020

All Science Journal Classification (ASJC) codes

  • Cell Biology

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