Abstract
PRMT5 is the primary enzyme responsible for the deposition of the symmetric dimethylarginine in mammalian cells. In an effort to understand how PRMT5 is regulated, we identified a threonine phosphorylation site within a C-terminal tail motif, which is targeted by the Akt/serum- and glucocorticoid-inducible kinases. While investigating the function of this posttranslational modification, we serendipitously discovered that its free C-terminal tail binds PDZ domains (when unphosphorylated) and 14-3-3 proteins (when phosphorylated). In essence, a phosphorylation event within the last few residues of the C-terminal tail generates a posttranslational modification-dependent PDZ/14-3-3 interaction "switch." The C-terminal motif of PRMT5 is required for plasma membrane association, and loss of this switching capacity is not compatible with life. This signaling phenomenon was recently reported for the HPV E6 oncoprotein but has not yet been observed for mammalian proteins. To investigate the prevalence of PDZ/14-3-3 switching in signal transduction, we built a protein domain microarray that harbors PDZ domains and 14-3-3 proteins. We have used this microarray to interrogate the C-terminal tails of a small group of candidate proteins and identified ERBB4, PGHS2, and IRK1 (as well as E6 and PRMT5) as conforming to this signaling mode, suggesting that PDZ/14-3-3 switching may be a broad biological paradigm.
Highlights
PRMT5 is the primary enzyme responsible for the deposition of the symmetric dimethylarginine in mammalian cells
We found that PRMT5 was both cytoplasmic and membrane-associated (Fig. 7C). 14-3-3 proteins predominantly associated with the cytoplasmic fraction, and NHERF2 predominantly associated with the membrane fraction
Possible Roles for PRMT5 on the Plasma Membrane—We found that PRMT5 associates with the plasma membrane and that this association is dependent on its C-terminal PDZ binding motif (Fig. 7)
Summary
PRMT5 Is Phosphorylated at Its C Terminus—Most arginine methyltransferases display robust intrinsic enzymatic activity in in vitro methylation assays [27], suggesting that in a cellular context, this enzymatic activity must be regulated, possibly by PTM of the PRMT enzymes themselves. The focused PDZ/14-3-3 microarray harbors 76 different mouse PDZ and all seven 14-3-3 proteins (Fig. 4) We probed this array with the PRMT5 peptide set, unmodified PRMT5(623– 637) or phosphorylated PRMT5pT634, and confirmed that the phosphopeptide bound 14-3-3 domains, whereas the unphosphorylated peptide bound several PDZ domains (Fig. 5, A and B). This allows a rough comparison of the relative strength of the different interactions, because the GST input can be well controlled (Fig. 6A, bottom) Using this approach, we found that the unphosphorylated tail of PRMT5 interacted most strongly with the GST fusion of full-length NHERF2, which harbors two PDZ domains. Of the seven PDZ domains identified as interacting with the C-terminal tail of PRMT5 (Fig. 5, A and B), six are associated with the plasma membrane, including the strongest binder, NHERF2. We were able to establish wild-type (4 clones) and heterozygous (6 clones) lines but not homozygous lines, which strongly suggests that the switching motif is required for the derivation of ES cells, as has been reported for PRMT5 null mice [40, 41]
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