DOK2 Inhibits EGFR-Mutated Lung Adenocarcinoma.
2013; 8 (11)
Oncogenic Tyrosine Kinases Target Dok-1 for Ubiquitin-Mediated Proteasomal Degradation To Promote Cell Transformation
MOLECULAR AND CELLULAR BIOLOGY
2011; 31 (13): 2552-2565
Somatic mutations in the EGFR proto-oncogene occur in ~15% of human lung adenocarcinomas and the importance of EGFR mutations for the initiation and maintenance of lung cancer is well established from mouse models and cancer therapy trials in human lung cancer patients. Recently, we identified DOK2 as a lung adenocarcinoma tumor suppressor gene. Here we show that genomic loss of DOK2 is associated with EGFR mutations in human lung adenocarcinoma, and we hypothesized that loss of DOK2 might therefore cooperate with EGFR mutations to promote lung tumorigenesis. We tested this hypothesis using genetically engineered mouse models and find that loss of Dok2 in the mouse accelerates lung tumorigenesis initiated by oncogenic EGFR, but not that initiated by mutated Kras. Moreover, we find that DOK2 participates in a negative feedback loop that opposes mutated EGFR; EGFR mutation leads to recruitment of DOK2 to EGFR and DOK2-mediated inhibition of downstream activation of RAS. These data identify DOK2 as a tumor suppressor in EGFR-mutant lung adenocarcinoma.
View details for DOI 10.1371/journal.pone.0079526
View details for PubMedID 24255704
The Rac activator DOCK7 regulates neuronal polarity through local phosphorylation of stathmin/Op18
2006; 51 (6): 727-739
Cellular transformation induced by oncogenic tyrosine kinases is a multistep process involving activation of growth-promoting signaling pathways and inactivation of suppressor molecules. Dok-1 is an adaptor protein that acts as a negative regulator of tyrosine kinase-initiated signaling and opposes oncogenic tyrosine kinase-mediated cell transformation. Findings that its loss facilitates transformation induced by oncogenic tyrosine kinases suggest that Dok-1 inactivation could constitute an intermediate step in oncogenesis driven by these oncoproteins. However, whether Dok-1 is subject to regulation by oncogenic tyrosine kinases remained unknown. In this study, we show that oncogenic tyrosine kinases, including p210(bcr-abl) and oncogenic forms of Src, downregulate Dok-1 by targeting it for degradation through the ubiquitin-proteasome pathway. This process is dependent on the tyrosine kinase activity of the oncoproteins and is mediated primarily by lysine-dependent polyubiquitination of Dok-1. Importantly, restoration of Dok-1 levels strongly suppresses transformation of cells expressing oncogenic tyrosine kinases, and this suppression is more pronounced in the context of a Dok-1 mutant that is largely refractory to oncogenic tyrosine kinase-induced degradation. Our findings suggest that proteasome-mediated downregulation of Dok-1 is a key mechanism by which oncogenic tyrosine kinases overcome the inhibitory effect of Dok-1 on cellular transformation and tumor progression.
View details for DOI 10.1128/MCB.05045-11
View details for Web of Science ID 000291431400002
View details for PubMedID 21536658
Dok-1 independently attenuates Ras/mitogen-activated protein kinase and Src/c-Myc pathways to inhibit platelet-derived growth factor-induced mitogenesis
MOLECULAR AND CELLULAR BIOLOGY
2006; 26 (7): 2479-2489
The polarization of a neuron generally results in the formation of one axon and multiple dendrites, allowing for the establishment of neuronal circuitry. The molecular mechanisms involved in priming one neurite to become the axon, particularly those regulating the microtubule network, remain elusive. Here we report the identification of DOCK7, a member of the DOCK180-related protein superfamily, as a Rac GTPase activator that is asymmetrically distributed in unpolarized hippocampal neurons and selectively expressed in the axon. Knockdown of DOCK7 expression prevents axon formation, whereas overexpression induces formation of multiple axons. We further demonstrate that DOCK7 and Rac activation lead to phosphorylation and inactivation of the microtubule destabilizing protein stathmin/Op18 in the nascent axon and that this event is important for axon development. Our findings unveil a pathway linking the Rac activator DOCK7 to a microtubule regulatory protein and highlight the contribution of microtubule network regulation to axon development.
View details for DOI 10.1016/j.neuron.2006.07.020
View details for Web of Science ID 000240997900012
View details for PubMedID 16982419
Lentiviral delivery of RNAi in hippocampal neurons
METHODS IN ENZYMOLOGY, VOL 406, REGULATORS AND EFFECTORS OF SMALL GTPASES: RHO FAMILY
2006; 406: 593-605
The Dok adaptor proteins play key regulatory roles in receptor and non-receptor kinase-initiated signaling pathways. Dok-1, the prototype member of this family, negatively regulates cell proliferation elicited by numerous growth factors, including platelet-derived growth factor (PDGF). However, how Dok-1 exerts its negative effect on mitogenesis has remained elusive. Using Dok-1 knockout cells and Dok-1 mutants deficient in binding to specific Dok-1-interacting proteins, we show that Dok-1 interferes with PDGF-stimulated c-myc induction and Ras/mitogen-activated protein kinase (MAPK) activation by tethering different signaling components to the cell membrane. Specifically, Dok-1 attenuates PDGF-elicited c-myc induction by recruiting Csk to active Src kinases, whereupon their activities and consequent c-myc induction are diminished. On the other hand, Dok-1 negatively regulates PDGF-induced MAPK activation by acting on Ras-GAP and at least one other Dok-1-interacting protein. Importantly, we demonstrate that Dok-1's actions on both of these signaling pathways contribute to its inhibitory effect on mitogenesis. Our data suggest a mechanistic basis for the inhibitory effect of Dok-1 on growth factor-induced mitogenesis and its role as a tumor suppressor.
View details for DOI 10.1128/MCB.26.7.2479-2489.2006
View details for Web of Science ID 000236312200002
View details for PubMedID 16537894
The breakthrough discovery that double-stranded RNA of 21 nucleotides in length (referred to as short or small interfering RNA; siRNA) can trigger sequence-specific gene silencing in mammalian cells has led to the development of a powerful new approach to study gene function (Dillon et al., 2005; Dykxhoorn et al., 2003; Elbashir et al., 2001; Hannon et al., 2004). Effective delivery of siRNA molecules into target cells or tissues is critical for successful RNA interference (RNAi) application. Here, we describe the use of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors for delivery of short hairpin RNA (shRNA), a precursor of siRNA, into primary neurons to suppress gene expression. Major advantages of lentiviral vectors are their ability to transduce nondividing cells and to confer long-term expression of transgenes. This chapter covers selection of short hairpin sequences, vector design, production of lentiviral supernatants, transduction of dissociated primary hippocampal neurons, and testing the effectiveness of shRNA-mediated silencing.
View details for DOI 10.1016/S0076-6879(06)06046-0
View details for Web of Science ID 000235750600046
View details for PubMedID 16472690