Transformation by the Rho-specific Guanine Nucleotide Exchange Factor Dbs Requires ROCK I-mediated Phosphorylation of Myosin Light Chain

Liu, Zhuoming; Костенко, Е. В.; Mahon, Gwendolyn M.; Olabisi, Oyenike O.; Whitehead, Ian P.

doi:10.1074/jbc.m601823200

Cited by 6 publications

(7 citation statements)

References 48 publications

(39 reference statements)

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“…Our previous studies have shown that onco-Dbs mediates transformation in fibroblasts through regulation of MLC2 in a RhoA/ROCK-dependent manner (37). Consistent with this, onco-Dbs activates RhoA when overexpressed in T47D cells.…”

Section: Discussionsupporting

confidence: 53%

“…Immunoprecipitation and Western Blot Analysis-Immunoprecipitation and Western blot analysis were performed as previously described (37). Antibodies used for Western blots Statistics-Statistical analyses were performed by one-way analysis of variance followed by the Tukey HSD (honestly significant difference) test for multiple comparisons using JMP statistical software.…”

Section: Methodsmentioning

confidence: 99%

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The Rho-specific Guanine Nucleotide Exchange Factor Dbs Regulates Breast Cancer Cell Migration

Liu

Adams

Whitehead

2009

Journal of Biological Chemistry

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Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that regulates neurotrophin-3-induced cell migration in Schwann cells. Here we report that Dbs regulates cell motility in tumor-derived, human breast epithelial cells through activation of Cdc42 and Rac1. Cdc42 and Rac1 are activated in T47D cells that stably express onco-or proto-Dbs, and activation is dependent upon growth of the cells on collagen I. Transient suppression of expression of Cdc42 or Rac1 by small interfering RNAs attenuates Dbs-enhanced motility. Both onco-and proto-Dbs-enhanced motility correlates with an increase in tyrosine phosphorylation of focal adhesion kinase on Tyr-397 and p130Cas on Tyr-410 and an increase in the abundance of the Crk⅐p130Cas complex. Suppression of expression of Cdc42 or its effector, Ack1, reduces tyrosine phosphorylation of focal adhesion kinase and p130Cas and disrupts the Crk⅐p130 Cas complex. We further determined that suppression of expression of Cdc42, Ack1, p130Cas , or Crk reduces Rac1 activation and cell motility in Dbs-expressing cells to a level comparable with that in vector cells. Therefore, a cascade of activation of Cdc42 and Rac1 by Dbs through the Cdc42 effector Ack1 and the Crk⅐p130Cas complex is established. Suppression of the expression of endogenous Dbs reduces cell motility in both T47D cells and MDA-MB-231 cells, which correlates with the down-regulation of Cdc42 activity. This suggests that Dbs activates Cdc42 in these two human breast cancer cell lines and that the normal function of Dbs may be required to support cell movement.Rho GTPases are a subfamily of the Ras superfamily of small signaling molecules that are widely expressed in mammalian cells (1). RhoA, Cdc42, and Rac1 are the most extensively studied members of the Rho GTPase family, and each plays a prominent and discrete role in cell migration (2, 3). Cdc42 promotes the formation of filopodia and is required to establish cell polarity (3-5); Rac1 promotes the formation of lamellipodia at the leading edge of motile cells (6), and RhoA promotes the formation of stress fibers which generate the traction forces needed to retract the cell tail and move the cell body beyond the leading edge (7,8). Consistent with this important role in cell motility, RhoA, Cdc42, and Rac1 are often overexpressed in human tumors including breast, lung, and colon (9), and overexpression of constitutively active RhoA, Cdc42, or Rac1 increases cell migration and invasion (2, 10, 11).The spatiotemporal regulation of Rho GTPase activity is tightly controlled by three classes of proteins. Rho-specific guanine nucleotide exchange factors (RhoGEFs) 2 activate Rho proteins by facilitating the exchange of GDP for GTP; Rho GTPaseactivating proteins (RhoGAPs) stimulate the intrinsic rate of hydrolysis of Rho proteins, thus converting them into their inactive state; Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) compete with RhoGEFs for binding to GDP-bound Rho proteins and sequester Rho in the inactive state (12).Dbs was identified in...

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Section: Discussionsupporting

confidence: 53%

Section: Methodsmentioning

confidence: 99%

The Rho-specific Guanine Nucleotide Exchange Factor Dbs Regulates Breast Cancer Cell Migration

Liu

Adams

Whitehead

2009

Journal of Biological Chemistry

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“…ACACA , RARA , NOTCH1 and NUP214 are known to form translocations in various types of hematological malignancies while many other fusion genes involve suspected oncogenes, such as RPS6KB1 ( RPS6KB1-TMEM49 and RPS6KB1-SNF8 ) [25], GSDMB ( TATDN1-GSDMB ) [26] and MCF2L ( LAMP1-MCF2L ) [27]. …”

Section: Discussionmentioning

confidence: 99%

Identification of fusion genes in breast cancer by paired-end RNA-sequencing

et al. 2011

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BackgroundUntil recently, chromosomal translocations and fusion genes have been an underappreciated class of mutations in solid tumors. Next-generation sequencing technologies provide an opportunity for systematic characterization of cancer cell transcriptomes, including the discovery of expressed fusion genes resulting from underlying genomic rearrangements.ResultsWe applied paired-end RNA-seq to identify 24 novel and 3 previously known fusion genes in breast cancer cells. Supported by an improved bioinformatic approach, we had a 95% success rate of validating gene fusions initially detected by RNA-seq. Fusion partner genes were found to contribute promoters (5' UTR), coding sequences and 3' UTRs. Most fusion genes were associated with copy number transitions and were particularly common in high-level DNA amplifications. This suggests that fusion events may contribute to the selective advantage provided by DNA amplifications and deletions. Some of the fusion partner genes, such as GSDMB in the TATDN1-GSDMB fusion and IKZF3 in the VAPB-IKZF3 fusion, were only detected as a fusion transcript, indicating activation of a dormant gene by the fusion event. A number of fusion gene partners have either been previously observed in oncogenic gene fusions, mostly in leukemias, or otherwise reported to be oncogenic. RNA interference-mediated knock-down of the VAPB-IKZF3 fusion gene indicated that it may be necessary for cancer cell growth and survival.ConclusionsIn summary, using RNA-sequencing and improved bioinformatic stratification, we have discovered a number of novel fusion genes in breast cancer, and identified VAPB-IKZF3 as a potential fusion gene with importance for the growth and survival of breast cancer cells.

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“…Construction of pAX142-IκB(SS), pAX142-H-ras-61L, pAX142-bcr(491-876) and pAX142-Tsg101 have been described previously [22,23]. pCAG-rock(KD) which encodes the kinase-defective version of ROCK I and pCAG-rock(WT) constructs were provided by S. Narumiya [24]. The reporter constructs utilized in the luciferase-coupled transcriptional assays, have been described previously [22,25].…”

Section: Molecular Constructsmentioning

confidence: 99%

ROCK I-mediated activation of NF-κB by RhoB

Rodrı́guez

Sahay

Olabisi

et al. 2007

Cellular Signalling

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RhoB is a short-lived protein whose expression is increased by a variety of extra-cellular stimuli including UV irradiation, epidermal growth factor (EGF) and transforming growth factor β (TGF-β). Whereas most Rho proteins are modified by the covalent attachment of a geranylgeranyl group, RhoB is unique in that it can exist in either a geranylgeranylated (RhoB-GG) or a farnesylated (RhoB-F) form. Although each form is proposed to have different cellular functions, the signaling events that underlie these differences are poorly understood. Here we show that RhoB can activate NF-κB signaling in multiple cell types. Whereas RhoB-F is a potent activator of NF-κB, much weaker activation is observed for RhoB-GG, RhoA, and RhoC. NF-κB activation by RhoB is not associated with increased nuclear translocation of RelA/p65, but rather, by modification of the RelA/p65 transactivation domain. Activation of NF-κB by RhoB is dependent upon ROCK I but not PRK I. Thus, ROCK I cooperates with RhoB to activate NF-κB, and suppression of ROCK I activity by genetic or pharmacological inhibitors blocks NF-κB activation. Suppression of RhoB activity by dominant-inhibitory mutants, or siRNA, blocks NF-κB activation by Bcr, and TSG101, but not by TNFα or oncogenic Ras. Collectively, these observations suggest the existence of an endosomeassociated pathway for NF-κB activation that is preferentially regulated by the farnesylated form of RhoB.

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Transformation by the Rho-specific Guanine Nucleotide Exchange Factor Dbs Requires ROCK I-mediated Phosphorylation of Myosin Light Chain

Cited by 6 publications

References 48 publications

The Rho-specific Guanine Nucleotide Exchange Factor Dbs Regulates Breast Cancer Cell Migration

The Rho-specific Guanine Nucleotide Exchange Factor Dbs Regulates Breast Cancer Cell Migration

Identification of fusion genes in breast cancer by paired-end RNA-sequencing

ROCK I-mediated activation of NF-κB by RhoB

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