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

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.

A dynein motor attachment complex regulates TGFß/Smad3 signaling.

Our previous results have demonstrated that km23-2 has functions in TGFß signaling that are distinct from those for km23-1. In the current report, we demonstrate that blockade of km23-2 decreased TGFß activation of the human Smad7 promoter Smad7-Luc, an endogenous Smad3-target promoter. Luminescence-based mammalian interaction mapping (LUMIER) analyses showed that TGFß stimulated the interaction of km23-2 preferentially with Smad3, relative to that with Smad2.

Decreased tumor progression and invasion by a novel anti-cell motility target for human colorectal carcinoma cells.

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.

Requirement for protein kinase A in the phosphorylation of the TGFβ receptor-interacting protein km23-1 as a component of TGFβ downstream effects.

km23-1 was previously identified as a TGFβ-receptor interacting protein that was phosphorylated on serines after TGFβ stimulation. In the current report, we examined the role of km23-1 phosphorylation in the downstream effects of TGFβ/protein kinase A (PKA) signaling. Using phosphorylation site prediction software, we found that km23-1 has two potential PKA consensus phosphorylation sites.

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

km23-1 was originally identified as a TGFß receptor-interacting protein that plays an important role in TGFß 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).

The TGFβ receptor-interacting protein km23-1/DYNLRB1 plays an adaptor role in TGFβ1 autoinduction via its association with Ras.

We have previously elucidated the signaling events that are required for TGFβ1 autoinduction (Yue, J., and Mulder, K. M.

Requirement of a dynein light chain in transforming growth factor β signaling in zebrafish ovarian follicle cells.

We have previously reported that the dynein light chains km23-1 and km23-2 are required for TGFβ signaling in mammalian cells. Here we describe another member of the km23/DYNLRB/LC7/robl family of dynein light chains in zebrafish, termed zkm23, which is also involved in TGFβ signaling. zkm23 was rapidly phosphorylated after TGFβ stimulation.

Overexpression of the dynein light chain km23-1 in human ovarian carcinoma cells inhibits tumor formation in vivo and causes mitotic delay at prometaphase/metaphase.

km23-1 is a dynein light chain that was identified as a TGFβ receptor-interacting protein. To investigate whether km23-1 controls human ovarian carcinoma cell (HOCC) growth, we established a tet-off inducible expression system in SKOV-3 cells in which the expression of km23-1 is induced upon doxycycline removal. We found that forced expression of km23-1 inhibited both anchorage-dependent and anchorage-independent growth of SKOV-3 cells.

Knockdown of c-Fos suppresses the growth of human colon carcinoma cells in athymic mice.

Here we have investigated whether inhibition of c-Fos expression in RKO human colon carcinoma cells (HCCCs) would result in reduced TGFβ1 expression and suppression of tumor growth in athymic mice. We stably transfected RKO cells with c-Fos small interfering RNA (siRNA) or with the corresponding control siRNA. Using these stable cell lines, we demonstrated that siRNA-c-Fos significantly suppressed both AP-1 binding, promoter reporter activity at the proximal AP-1 site in the TGFβ1 promoter, and TGFβ1 production.

Requirement of a dynein light chain in TGFbeta/Smad3 signaling.

We have previously reported that the dynein light chain (DLC) km23-1 is required for Smad2-dependent TGFbeta signaling. Here we describe another member of the km23/DYNLRB/LC7/robl family of DLCs, termed km23-2, which is also involved in TGFbeta signaling. We show not only that TGFbeta stimulates the interaction of km23-2 (DYNLRB2) with TGFbeta receptor II (TbetaRII) but also that TGFbeta regulates the interaction between km23-2 and endogenous TbetaRII in vivo.

Requirement for the dynein light chain km23-1 in a Smad2-dependent transforming growth factor-beta signaling pathway.

We have identified km23-1 as a novel transforming growth factor-beta (TGFbeta) receptor (TbetaR)-interacting protein that is also a light chain of the motor protein dynein (dynein light chain). Herein, we demonstrate by sucrose gradient analyses that, in the presence of TGFbeta but not in the absence, km23-1 was present in early endosomes with the TbetaRs. Further, confocal microscopy studies indicate that endogenous km23-1 was co-localized with endogenous Smad2 at early times after TGFbeta treatment, prior to Smad2 translocation to the nucleus.

Requirement of Smad3 and CREB-1 in mediating transforming growth factor-beta (TGF beta) induction of TGF beta 3 secretion.

Because increased transforming growth factor-beta (TGFbeta) production by tumor cells contributes to cancer progression through paracrine mechanisms, identification of critical points that can be targeted to block TGFbeta production is important. Previous studies have identified the precise signaling components and promoter elements required for TGFbeta induction of TGFbeta1 expression in epithelial cells (Yue, J., and Mulder, K.

c-Fos is required for TGFbeta1 production and the associated paracrine migratory effects of human colon carcinoma cells.

In tumor cells that have lost responsiveness to the growth inhibitory effects of transforming growth factor beta (TGFbeta), increased TGFbeta production by the tumor cells often contributes to cancer progression, primarily through paracrine mechanisms. Here we investigated the major components of the activator protein-1 (AP-1) complex in the TGFbeta1 promoter of human colon carcinoma cells (HCCCs). In contrast to untransformed epithelial cells (UECs), HCCCs displayed constitutive activation of AP-1 at the proximal AP-1 site in the human TGFbeta1 promoter.

Structure and dynamics of the homodimeric dynein light chain km23.

km23 (96 residues, 11 kDa) is the mammalian ortholog of Drosophila roadblock, the founding member of LC7/robl/km23 class of dynein light chains. km23 has been shown to be serine-phosphorylated following TGFbeta receptor activation and to bind the dynein intermediate chain in response to such phosphorylation. Here, we report the three-dimensional solution structure of km23, which is shown to be that of a homodimer, similar to that observed for the heterodimeric complex formed between p14 and MP1, two distantly related members of the MglB/robl superfamily, but distinct from the LC8 and Tctex-1 classes of dynein light chains, which also adopt homodimeric structures.

A transforming growth factor-beta receptor-interacting protein frequently mutated in human ovarian cancer.

Ovarian carcinomas, particularly recurrent forms, are frequently resistant to transforming growth factor-beta (TGF-beta)-mediated growth inhibition. However, mutations in the TGF-beta receptor I and receptor II (TbetaR-I and TbetaR-II) genes have only been reported in a minority of ovarian carcinomas, suggesting that alterations in TGF-beta-signaling components may play an important role in the loss of TGF-beta responsiveness. Using laser-capture microdissection and nested reverse-transcription-PCR, we found that km23, which interacts with the TGF-beta receptor complex, is altered at a high frequency in human ovarian cancer patients.

Requirement of km23 for TGFbeta-mediated growth inhibition and induction of fibronectin expression.

We previously identified km23 as a novel TGFbeta receptor-interacting protein. Here we show that km23 is ubiquitously expressed in human tissues and that cell-type specific differences in endogenous km23 protein expression exist. In addition, we demonstrate that the phosphorylation of km23 is TGFbeta-dependent, in that EGF was unable to phosphorylate km23.

Requirement of TGF-beta receptor-dependent activation of c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases (Sapks) for TGF-beta up-regulation of the urokinase-type plasminogen activator receptor.

We have previously demonstrated that activation of the Ras/Mapk pathways is required for transforming growth factor beta (TGF-beta) induction of TGF-beta(1) expression. Here we examined the role of the Ras/Mapk pathways in TGF-beta induction of urokinase-type plasminogen activator receptor (uPAR) expression in untransformed intestinal epithelial cells (IECs). TGF-beta activated the stress-activated protein kinases (Sapk)/c-Jun N-terminal kinases (JNKs) within 5-10 min, an effect that preceeded TGF-beta induction of uPAR expression in these cells.

A novel transforming growth factor-beta receptor-interacting protein that is also a light chain of the motor protein dynein.

The phosphorylated, activated cytoplasmic domains of the transforming growth factor-beta (TGFbeta) receptors were used as probes to screen an expression library that was prepared from a highly TGFbeta-responsive intestinal epithelial cell line. One of the TGFbeta receptor-interacting proteins isolated was identified to be the mammalian homologue of the LC7 family (mLC7) of dynein light chains (DLCs). This 11-kDa cytoplasmic protein interacts with the TGFbeta receptor complex intracellularly and is phosphorylated on serine residues after ligand-receptor engagement.