pseudosubstrate sequence
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2018 ◽  
Author(s):  
Xueyin Wang ◽  
Yicheng Long ◽  
Richard D. Paucek ◽  
Anne R. Gooding ◽  
Thomas Lee ◽  
...  

ABSTRACTPolycomb Repressive Complex 2 (PRC2) is a histone methyltransferase whose function is critical for regulating transcriptional repression in many eukaryotes including humans. Its catalytic moiety EZH2 is responsible for the tri-methylation of H3K27 and also undergoes automethylation. Using mass spectroscopic analysis of recombinant human PRC2, we identified three methylated lysine residues (K510, K514, K515) on a disordered but highly conserved loop of EZH2. These lysines were mostly mono- and di-methylated. Either mutation of these lysines or their methylation increases PRC2 histone methyltransferase activity. In addition, mutation of these three lysines in HEK293T cells using CRISPR genome-editing increases global H3K27 methylation levels. EZH2 automethylation occurs intramolecularly (in cis) by methylation of a pseudosubstrate sequence on the flexible loop. This post-translational modification andcis-regulation of PRC2 are analogous to the activation of many protein kinases by autophosphorylation. We therefore propose that EZH2 automethylation provides a way for PRC2 to modulate its histone methyltransferase activity by sensing histone H3 tails, SAM concentration, and perhaps other effectors.


2007 ◽  
Vol 26 (3) ◽  
pp. 806-815 ◽  
Author(s):  
John W Scott ◽  
Fiona A Ross ◽  
J K David Liu ◽  
D Grahame Hardie

2003 ◽  
Vol 373 (2) ◽  
pp. 571-581 ◽  
Author(s):  
Angie F. KIRWAN ◽  
Ashley C. BIBBY ◽  
Thierry MVILONGO ◽  
Heimo RIEDEL ◽  
Thomas BURKE ◽  
...  

The N-terminal pseudosubstrate site within the protein kinase Cα (PKCα)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCα-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940–8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCα-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Rα39–177: this protein contained the full regulatory domain of human PKCα fused to glutathione S-transferase (GST), but lacked amino acids 1–38 (including the pseudosubstrate sequence) and amino acids 178–270 (including the C2 region)]. GST-Rα39–177 significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCα holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33–86 within GST-Rα39–177 dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159–242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of multiple regions within the PKCα-regulatory domain that play a direct role in the inhibition of catalytic domain activity.


1996 ◽  
Vol 271 (34) ◽  
pp. 20748-20755 ◽  
Author(s):  
Sharron H. Francis ◽  
Jeffrey A. Smith ◽  
Janet L. Colbran ◽  
Kennard Grimes ◽  
Kenneth A. Walsh ◽  
...  

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