ABSTRACT
Death receptor-dependent apoptosis is an important mechanism of growth control. It has been demonstrated that Ras association domain family protein 1A (RASSF1A) is a tumor suppressor protein involved in death receptor-dependent apoptosis. However, it is unclear how RASSF1A-mediated cell death is initiated. We have now detailed 14-3-3 dependent regulation of RASSF1A-mediated cell death. We demonstrate that basal association of RASSF1A with 14-3-3 was lost following stimulation with tumor necrosis factor alpha (TNFalpha) or TNFalpha related apoptosis inducing ligand (TRAIL). Subsequent to the loss of 14-3-3 association, RASSF1A associated with modulator of apoptosis (MOAP-1) followed by death receptor association with either TNFalpha receptor 1 (TNF-R1) or TRAIL receptor 1 (TRAIL-R1). 14-3-3 association required basal phosphorylation by the serine/threonine kinase, glycogen synthase kinase 3beta (GSK-3beta), on serine 175, 178, and 179. Mutation of these critical serines resulted in the loss of 14-3-3 association and earlier recruitment of RASSF1A to MOAP-1, TNF-R1, and TRAIL-R1. Furthermore, stable cells containing a triple serine mutant of RASSF1A [serine (S) 175 to alanine (A) [S175A], S178A, and S179A] resulted in increased basal cell death, enhanced Annexin V staining and enhanced cleavage of poly (ADP-ribose) polymerase (PARP) following TNFalpha stimulation when compared to stable cells containing wild type RASSF1A. RASSF1A-mediated cell death is, therefore, tightly controlled by 14-3-3 association.
Subject(s)
14-3-3 Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Apoptosis Regulatory Proteins/metabolism , Binding Sites , Cell Death , Cell Line, Tumor , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Kinetics , Models, Biological , Molecular Sequence Data , Mutant Proteins/metabolism , Phosphorylation , Protein Binding , Receptors, Death Domain/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Suppressor Proteins/chemistryABSTRACT
Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a(+/-) , Rassf1a(-/-) and an intestinal epithelial cell specific knockout mouse (Rassf1a (IEC-KO) ) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a(-/-) mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a(-/-) background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a(-/-) mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.
Subject(s)
Colitis, Ulcerative/genetics , Colon/metabolism , Epithelial Cells/metabolism , Intestinal Mucosa/metabolism , NF-kappa B/genetics , Toll-Like Receptors/genetics , Tumor Suppressor Proteins/genetics , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis/drug effects , Benzamides/pharmacology , Cell Cycle Proteins , Cell Proliferation/drug effects , Colitis, Ulcerative/chemically induced , Colitis, Ulcerative/drug therapy , Colitis, Ulcerative/pathology , Colon/drug effects , Colon/pathology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dextran Sulfate , Epithelial Cells/drug effects , Epithelial Cells/pathology , Gene Expression Regulation , Imatinib Mesylate , Inflammation/chemically induced , Inflammation/drug therapy , Inflammation/genetics , Inflammation/pathology , Intestinal Mucosa/drug effects , Intestinal Mucosa/pathology , Mice , Mice, Knockout , NF-kappa B/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Piperazines/pharmacology , Proto-Oncogene Proteins c-abl/pharmacology , Pyrimidines/pharmacology , Signal Transduction , Toll-Like Receptors/metabolism , Tumor Protein p73 , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/metabolism , YAP-Signaling Proteins , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
RASSF1A is a tumor suppressor protein involved in death receptor-dependent apoptosis utilizing the Bax-interacting protein MOAP-1 (previously referred to as MAP-1). However, the dynamics of death receptor recruitment of RASSF1A and MOAP-1 are still not understood. We have now detailed recruitment to death receptors (tumor necrosis factor receptor 1 [TNF-R1] and TRAIL-R1/DR4) and identified domains of RASSF1A and MOAP-1 that are required for death receptor interaction. Upon TNF-alpha stimulation, the C-terminal region of MOAP-1 associated with the death domain of TNF-R1; subsequently, RASSF1A was recruited to MOAP-1/TNF-R1 complexes. Prior to recruitment to TNF-R1/MOAP-1 complexes, RASSF1A homodimerization was lost. RASSF1A associated with the TNF-R1/MOAP-1 or TRAIL-R1/MOAP-1 complex via its N-terminal cysteine-rich (C1) domain containing a potential zinc finger binding motif. Importantly, TNF-R1 association domains on both MOAP-1 and RASSF1A were essential for death receptor-dependent apoptosis. The association of RASSF1A and MOAP-1 with death receptors involves an ordered recruitment to receptor complexes to promote cell death and inhibit tumor formation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , Receptors, Death Domain/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Apoptosis , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Humans , Protein Interaction Domains and Motifs , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Signal Transduction , Tumor Suppressor Proteins/chemistryABSTRACT
Calcineurin was demonstrated to regulate the phosphorylation of threonine (T)-172 of CDK4. We further investigated how calcineurin can regulate this essential post-translational modification on CDK4. In this study, we demonstrate that calcineurin can associate predominantly with the cytoplasmic form of CDK4 in the absence of cyclin D. The inhibition of calcineurin phosphatase activity resulted in the specific increase of the phosphorylation and activity levels of CDK4 within the mitotic fraction. The association of calcineurin with CDK4 peaked during the mitotic phase of the cell cycle and coincided with reduction of CDK4 phosphorylation. Using structural mutants to CDK4, we localized the interaction site of calcineurin within the amino terminal residues of CDK4 that are important for both cyclin D and p16INK4a binding. Our data suggest that calcineurin may regulate the kinase activity of CDK4 in a cell cycle-dependent manner and may be an important component of the negative regulation of CDK4.