Browsing by Subject "Substrate selectivity"

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    Autoregulatory and structural control of CaMKII substrate specificity
    (2016-09) Johnson, Derrick Ethan; Hudmon, Andy; Hurley, Thomas D.; Hoang, Quyen Q.; Gallagher, Patricia
    Calcium/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a multimeric holoenzyme composed of 8–14 subunits from four closely related isoforms (α, β, γ, δ). CaMKII plays a strategic, multifunctional role in coupling the universal second messenger calcium with diverse cellular processes including metabolism, cell cycle control, and synaptic plasticity. CaMKII exhibits broad substrate specificity, targeting numerous substrates with diverse phosphorylation motifs. Binding of the calcium sensor CaM to the autoregulatory domain (ARD) of CaMKII functions to couple kinase activation with calcium signaling. Important sites of autophosphorylation, namely T287 and T306/7 (δ isoform numbering), reside within the ARD and control either CaM dependence or ability to bind to CaMKII respectively, thus determining various activation states of the kinase. Because autophosphorylation is critical to the function of CaMKII in vivo, we sought to determine the relationship between the activation state of the kinase and substrate selectivity. We show that the ARD of activated CaMKII tunes substrate selectivity by competing for substrate binding to the catalytic domain, thus functioning as a selectivity filter. Specifically, in the absence of T287 autophosphorylation, substrate phosphorylation is limited to high-affinity, consensus substrates. T287 autophosphorylation restores maximal kinase activation and broad substrate selectivity by disengaging ARD filtering. The unique multimeric architecture of CaMKII is an ideal sensor which encodes calcium-spike frequency into graded levels of subunit activation/autophosphorylation within the holoenzyme. We find that differential activation states of the holoenzyme produce distinct substrate phosphorylation profiles. Maximal holoenzyme activation/autophosphorylation leads to further broadening of substrate specificity beyond the effect of autophosphorylation alone, which is consistent with multivalent avidity. Thus, the ability of calcium-spike frequency to regulate T287 autophosphorylation and holoenzyme activation permits cellular activity to dictate switch-like behavior in substrate selectivity that is required for diverse cellular responses by CaMKII.
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    Identification of nonhistone substrates of the lysine methyltransferase PRDM9
    (Elsevier, 2023) Hanquier, Jocelyne N.; Sanders, Kenidi; Berryhill, Christine A.; Sahoo, Firoj K.; Hudmon, Andy; Vilseck, Jonah Z.; Cornett, Evan M.; Biochemistry and Molecular Biology, School of Medicine
    Lysine methylation is a dynamic, posttranslational mark that regulates the function of histone and nonhistone proteins. Many of the enzymes that mediate lysine methylation, known as lysine methyltransferases (KMTs), were originally identified to modify histone proteins but have also been discovered to methylate nonhistone proteins. In this work, we investigate the substrate selectivity of the KMT PRDM9 to identify both potential histone and nonhistone substrates. Though normally expressed in germ cells, PRDM9 is significantly upregulated across many cancer types. The methyltransferase activity of PRDM9 is essential for double-strand break formation during meiotic recombination. PRDM9 has been reported to methylate histone H3 at lysine residues 4 and 36; however, PRDM9 KMT activity had not previously been evaluated on nonhistone proteins. Using lysine-oriented peptide libraries to screen potential substrates of PRDM9, we determined that PRDM9 preferentially methylates peptide sequences not found in any histone protein. We confirmed PRDM9 selectivity through in vitro KMT reactions using peptides with substitutions at critical positions. A multisite λ-dynamics computational analysis provided a structural rationale for the observed PRDM9 selectivity. The substrate selectivity profile was then used to identify putative nonhistone substrates, which were tested by peptide spot array, and a subset was further validated at the protein level by in vitro KMT assays on recombinant proteins. Finally, one of the nonhistone substrates, CTNNBL1, was found to be methylated by PRDM9 in cells.