A majority of the 90 human protein tyrosine kinases (PTKs) are understudied "orphan" enzymes with few or no known substrates. Designing experiments aimed at assaying the catalytic activity of these PTKs has been a long-running problem. In the past, researchers have used polypeptides with a randomized 4:1 molar ratio of glutamic acid to tyrosine as general PTK substrates. However, these substrates are inefficient and perform poorly for many applications. In this work, we apply the KINATEST-ID pipeline for artificial kinase substrate discovery to design a set of candidate "universal" PTK peptide substrate sequences. We identified two unique peptide sequences from this set that had robust activity with a panel of 15 PTKs tested in an initial screen. Kinetic characterization with seven receptor and nonreceptor PTKs confirmed these peptides to be efficient and general PTK substrates. The broad scope of these artificial substrates demonstrates that they should be useful as tools for probing understudied PTK activity.
PDB Reference: casein kinase 1 , 4jjrCasein kinase 1 (CK1) is a regulatory enzyme in the mammalian circadian oscillator and represents a potential pharmacological target for modulating circadian rhythms. Crystal structures of four different polymorphs of CK1 have previously been determined and this article reports the crystallization and structure determination of a new crystal form belonging to space group P2 1 . Comparison of CK1 crystal structures reveals few conformational differences within the C-terminal lobe, but more significant movements of the -sheet region of the N-terminal lobe were observed.
Casein kinase 1ε (CK1ε) plays an important regulatory role in various cellular processes including circadian rhythms. Mutations in CK1ε or the recognition site on its substrate PER2 result in modulation of the circadian period length. In particular, the tau mutation (R178C) in the catalytic domain of CK1ε was identified as the molecular basis for a dose-dependent heritable shortened circadian period in hamsters. However, the biochemical basis for the physiological effects of the tau mutant remains unclear. It has been reported that the tau mutation has reduced in vitro activity against some substrates but increased in vitro activity against other substrates. To better understand the effects of the CK1ε tau mutation, an ATP-phosphopeptide conjugate was synthesized to yield a transition-state bisubstrate analog. Kinase activity assays determined that the tau mutant has 80% reduced activity and a fourfold decrease in sensitivity to the bisubstrate analog compared to wild type. This confirms that Arg178 is important in the recognition of the preferred phosphosubstrates of CK1ε.
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