Small molecule inhibitors and CRISPR/Cas9 mutagenesis demonstrate that SMYD2 and SMYD3 activity are dispensable for autonomous cancer cell proliferation.

A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed.

CARM1 Preferentially Methylates H3R17 over H3R26 through a Random Kinetic Mechanism.

CARM1 is a type I arginine methyltransferase involved in the regulation of transcription, pre-mRNA splicing, cell cycle progression, and the DNA damage response. CARM1 overexpression has been implicated in breast, prostate, and liver cancers and therefore is an attractive target for cancer therapy. To date, little about the kinetic properties of CARM1 is known.

Characterization of the Enzymatic Activity of SETDB1 and Its 1:1 Complex with ATF7IP.

The protein methyltransferase (PMT) SETDB1 is a strong candidate oncogene in melanoma and lung carcinomas. SETDB1 methylates lysine 9 of histone 3 (H3K9), utilizing S-adenosylmethionine (SAM) as the methyl donor and its catalytic activity, has been reported to be regulated by a partner protein ATF7IP. Here, we examine the contribution of ATF7IP to the in vitro activity and substrate specificity of SETDB1.

Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras.

Role of the histone domain in the autoinhibition and activation of the Ras activator Son of Sevenless.

Membrane-bound Ras is activated by translocation of the Son of Sevenless (SOS) protein to the plasma membrane. SOS is inactive unless Ras is bound to an allosteric site on SOS, and the Dbl homology (DH) and Pleckstrin homology (PH) domains of SOS (the DH-PH unit) block allosteric Ras binding. We showed previously that the activity of SOS at the membrane increases with the density of PIP(2) and the local concentration of Ras-GTP, which synergize to release the DH-PH unit.

Membrane-dependent signal integration by the Ras activator Son of sevenless.

The kinetics of Ras activation by Son of sevenless (SOS) changes profoundly when Ras is tethered to membranes, instead of being in solution. SOS has two binding sites for Ras, one of which is an allosteric site that is distal to the active site. The activity of the SOS catalytic unit (SOS(cat)) is up to 500-fold higher when Ras is on membranes compared to rates in solution, because the allosteric Ras site anchors SOS(cat) to the membrane.

An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor.

The mechanism by which the epidermal growth factor receptor (EGFR) is activated upon dimerization has eluded definition. We find that the EGFR kinase domain can be activated by increasing its local concentration or by mutating a leucine (L834R) in the activation loop, the phosphorylation of which is not required for activation. This suggests that the kinase domain is intrinsically autoinhibited, and an intermolecular interaction promotes its activation.

Calcium-independent stimulation of membrane fusion and SNAREpin formation by synaptotagmin I.

Neurotransmitter release requires the direct coupling of the calcium sensor with the machinery for membrane fusion. SNARE proteins comprise the minimal fusion machinery, and synaptotagmin I, a synaptic vesicle protein, is the primary candidate for the main neuronal calcium sensor. To test the effect of synaptotagmin I on membrane fusion, we incorporated it into a SNARE-mediated liposome fusion assay.