ABSTRACT
TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor-activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named "TRP channel-associated factors" (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity.
Subject(s)
Adenocarcinoma/metabolism , Membrane Proteins/metabolism , Prostate/metabolism , Prostatic Neoplasms/metabolism , TRPM Cation Channels/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Amino Acid Sequence , Animals , Cell Line, Tumor , Cell Movement , HEK293 Cells , Humans , Ion Channel Gating , Kinetics , Male , Membrane Potentials , Membrane Proteins/genetics , Mice, Inbred C57BL , Middle Aged , Molecular Sequence Data , Neoplasm Invasiveness , Prostate/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Protein Binding , Protein Transport , RNA Interference , Signal Transduction , TRPM Cation Channels/genetics , TransfectionABSTRACT
PEA3, ERM and ER81 belong to the PEA3 subfamily of Ets transcription factors and play important roles in a number of tissue-specific processes. Transcriptional activation by PEA3 subfamily factors requires their characteristic amino-terminal acidic transactivation domain (TAD). However, the cellular targets of this domain remain largely unknown. Using ERM as a prototype, we show that the minimal N-terminal TAD activates transcription by contacting the activator interacting domain (ACID)/Prostate tumor overexpressed protein 1 (PTOV) domain of the Mediator complex subunit MED25. We further show that depletion of MED25 disrupts the association of ERM with the Mediator in vitro. Small interfering RNA-mediated knockdown of MED25 as well as the overexpression of MED25-ACID and MED25-VWA domains efficiently inhibit the transcriptional activity of ERM. Moreover, mutations of amino acid residues that prevent binding of MED25 to ERM strongly reduce transactivation by ERM. Finally we show that siRNA depletion of MED25 diminishes PEA3-driven expression of MMP-1 and Mediator recruitment. In conclusion, this study identifies the PEA3 group members as the first human transcriptional factors that interact with the MED25 ACID/PTOV domain and establishes MED25 as a crucial transducer of their transactivation potential.
Subject(s)
DNA-Binding Proteins/metabolism , Mediator Complex/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Cell Line , DNA-Binding Proteins/chemistry , Humans , Mediator Complex/chemistry , Mediator Complex/genetics , Mutation , Protein Interaction Domains and Motifs , Transcription Factors/chemistryABSTRACT
The PEA3 (polyoma enhancer activator 3) group members [ERM (ETS-related molecule), ER81 (ETS-related 81) and PEA3] of the Ets transcription factor family are involved in migration and dissemination processes during organogenesis and cancer development. In the present study, we report that the hnRNP (heterogeneous nuclear ribonucleoprotein)-like protein CoAA (Coactivator activator) interacts with the PEA3 group members and modulates their transcriptional activity. We also demonstrate that the CoAA YQ domain, containing tyrosine/glutamine-rich hexapeptide repeats, is necessary for the interaction, whereas the two N-terminal RRMs (RNA recognition motifs) of CoAA are required to enhance transcriptional activity. Finally, we show that CoAA is involved in the migration-enhancing action of PEA3 on MCF7 human cancer cells, suggesting that CoAA might be an important regulator of PEA3 group member activity during metastasis.
Subject(s)
Intracellular Signaling Peptides and Proteins/physiology , Transcription Factors/biosynthesis , Transcriptional Activation/physiology , Animals , Cell Movement/genetics , HEK293 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Rabbits , Transcription Factors/geneticsABSTRACT
Erm, Er81, and Pea3 are the three members of the PEA3 group which belong to the Ets transcription factors family. These proteins regulate transcription of multiple target genes, such as those encoding several matrix metalloproteinases (MMP), which are enzymes degrading the extracellular matrix during cancer metastasis. In fact, PEA3-group genes are often overexpressed in different types of human cancers that also over-express these MMP and display a disseminating phenotype. In experimental models, regulation of PEA3 group member expression has been shown to influence the metastatic process, thus suggesting that these factors play a key role in metastasis.
Subject(s)
Matrix Metalloproteinases/metabolism , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Rearrangement/genetics , Humans , Mice , Neoplasm Metastasis , Neoplasm Proteins/genetics , Neoplasms/pathology , Proto-Oncogene Proteins c-ets/genetics , Proto-Oncogene Proteins c-ets/metabolism , Transcription Factors/geneticsABSTRACT
The PEA3 group is composed of three highly conserved Ets transcription factors: Erm, Er81, and Pea3. These proteins regulate transcription of multiple genes, and their transactivating potential is affected by post-translational modifications. Among their target genes are several matrix metalloproteases (MMPs), which are enzymes degrading the extracellular matrix during normal remodelling events and cancer metastasis. In fact, PEA3-group genes are often over-expressed in different types of cancers that also over-express these MMPs and display a disseminating phenotype. Experimental regulation of the synthesis of PEA3 group members influences the metastatic process. This suggests that these factors play a key role in metastasis.
