The TGFβ Receptor-interacting Protein km23-1/DYNLRB1 Plays an Adaptor Role in TGFβ1 Autoinduction via Its Association with Ras

Jin, Qunyan; Ding, Wei; Mulder, Kathleen M.

doi:10.1074/jbc.m112.344887

Cited by 15 publications

(20 citation statements)

References 59 publications

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“…The pRNATin-H1.2/hygro km23-1 siRNA and the relevant NC siRNA sequences have been described previously [34], [36]. Further, we have developed RKO cell clones (#1, #5) stably expressing km23-1-specific siRNA and confirmed km23-1 depletion by Western blot analysis of protein lysates isolated from the stable transfectants as described previously [35]. We further confirmed the efficiency of km23-1 silencing in RKO cells at the mRNA level.…”

Section: Resultssupporting

confidence: 69%

“…These pRNATin-H1.2/hygro siRNA vectors were transfected into RKO cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The empty vector (EV), NC siRNA, and km23-1 siRNA (km23-1 siRNA clone #1 and clone #5) RKO stable transfectants were prepared, characterized, and routinely maintained in growth medium with 100 µg/ml of hygromycin B as described previously [35].…”

Section: Methodsmentioning

confidence: 99%

“…NMR and X-ray crystal structure analyses demonstrated that km23-1 exists in cells as a stable homodimer, structurally unrelated to other dynein light chains (Tctex1/DYNLT and LC8/DYNLL) [32], [33]. However, as for these other dynein light chain families, km23-1 is multifunctional and can regulate both dynein-dependent and -independent functions [34], [35]. Moreover, km23-1 has been shown to mediate specific TGFβ signaling events and downstream responses [31], [34], [36].…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, km23-1 has been shown to mediate specific TGFβ signaling events and downstream responses [31], [34], [36]. For example, km23-1 is required for TGFβ1 production through dynein-independent Ras/ERK/Jun N-terminal kinase (JNK) pathways in TGFβ-sensitive epithelial cells [35]. This multifunctional regulator also plays a critical dynein-dependent role in Smad2 signaling in early endosomes [34], and it is regulated by protein kinase A (PKA) activation [37].…”

Section: Introductionmentioning

confidence: 99%

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Decreased Tumor Progression and Invasion by a Novel Anti-Cell Motility Target for Human Colorectal Carcinoma Cells

et al. 2013

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We have previously described a novel modulator of the actin cytoskeleton that also regulates Ras and mitogen-activated protein kinase activities in TGFβ-sensitive epithelial cells. Here we examined the functional role of this signaling regulatory protein (km23-1) in mediating the migration, invasion, and tumor growth of human colorectal carcinoma (CRC) cells. We show that small interfering RNA (siRNA) depletion of km23-1 in human CRC cells inhibited constitutive extracellular signal-regulated kinase (ERK) activation, as well as pro-invasive ERK effector functions that include phosphorylation of Elk-1, constitutive regulation of c-Fos-DNA binding, TGFβ1 promoter transactivation, and TGFβ1 secretion. In addition, knockdown of km23-1 reduced the paracrine effects of CRC cell-secreted factors in conditioned medium and in fibroblast co-cultures. Moreover, km23-1 depletion in human CRC cells reduced cell migration and invasion, as well as expression of the ERK-regulated, metastasis-associated scaffold protein Ezrin. Finally, km23-1 inhibition significantly suppressed tumor formation in vivo. Thus, our results implicate km23-1 as a novel anti-metastasis target for human colon carcinoma cells, capable of decreasing tumor growth and invasion via a mechanism involving suppression of various pro-migratory features of CRC. These include a reduction in ERK signaling, diminished TGFβ1 production, decreased expression of the plasma membrane-cytoskeletal linker Ezrin, as well as attenuation of the paracrine effects of colon carcinoma-secreted factors on fibroblast migration and mitogenesis. As such, km23-1 inhibitors may represent a viable therapeutic strategy for interfering with colon cancer progression and invasion.

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Section: Resultssupporting

confidence: 69%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Decreased Tumor Progression and Invasion by a Novel Anti-Cell Motility Target for Human Colorectal Carcinoma Cells

et al. 2013

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“…More recently, we have shown that Ras and km23-1 form a TGFβ-regulated complex in vivo [17]. However, km23-1 is actually part of an ancient superfamily, widely represented in archaea and bacteria, which appears to be involved in regulating NTPase activity [18].…”

Section: Introductionmentioning

confidence: 99%

Role of km23-1 in RhoA/actin-based cell migration

Jin

Pulipati

Zhou

et al. 2012

Biochemical and Biophysical Research Communications

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km23-1 was originally identified as a TGFβ receptor-interacting protein that plays an important role in TGFβ signaling. Moreover, km23-1 is actually part of an ancient superfamily of NTPase-regulatory proteins, widely represented in archaea and bacteria. To further elucidate the function of km23-1, we identified novel protein interacting partners for km23-1 by using tandem affinity purification (TAP) and tandem mass spectrometry (MS). Here we show that km23-1 interacted with a class of proteins involved in actin-based cell motility and modulation of the actin cytoskeleton. We further showed that km23-1 modulates the formation of a highly organized stress fiber network. More significantly, we demonstrated that knockdown (KD) of km23-1 decreased RhoA activation in Mv1Lu epithelial cells. Finally, our results demonstrated for the first time that depletion of km23-1 inhibited cell migration of human colon carcinoma cells (HCCCs) in wound-healing assays. Overall, our findings demonstrate that km23-1 regulates RhoA and motility-associated actin modulating proteins, suggesting that km23-1 may represent a novel target for anti-metastatic therapy.

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

Raza

Pandey

Jin

et al. 2019

Cell Biology International

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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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The TGFβ Receptor-interacting Protein km23-1/DYNLRB1 Plays an Adaptor Role in TGFβ1 Autoinduction via Its Association with Ras

Cited by 15 publications

References 59 publications

Decreased Tumor Progression and Invasion by a Novel Anti-Cell Motility Target for Human Colorectal Carcinoma Cells

Decreased Tumor Progression and Invasion by a Novel Anti-Cell Motility Target for Human Colorectal Carcinoma Cells

Role of km23-1 in RhoA/actin-based cell migration

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

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