Defining the Substrate Specificity Determinants Recognized by the Active Site of C-Terminal Src Kinase-Homologous Kinase (CHK) and Identification of β-Synuclein as a Potential CHK Physiological Substrate

Abstract

C-terminal Src kinase-homologous kinase (CHK) exerts its tumor suppressor function by phosphorylating the C-terminal regulatory tyrosine of the Src-family kinases (SFKs). The phosphorylation suppresses their activity and oncogenic action. In addition to phosphorylating SFKs, CHK also performs non-SFK related functions by phosphorylating other cellular protein substrates. To define these non-SFK related functions of CHK, we used the `kinase substrate tracking and elucidation' method to search for its potential physiological substrates in rat brain cytosol. Our search revealed β-synuclein as a potential CHK substrate, and Y127 in β-synuclein as the preferential phosphorylation site. Using peptides derived from β-synuclein and positional scanning combinatorial peptide library screening, we defined the optimal substrate phosphorylation sequence recognized by the CHK active site to be E-x-[Φ/E/D]-Y-Φ-x-Φ, where Φ and x represent hydrophobic residues and any residue, respectively. Besides β-synuclein, cellular proteins containing motifs resembling this sequence are potential CHK substrates. Intriguingly, the CHK-optimal substrate phosphorylation sequence bears little resemblance to the C-terminal tail sequence of SFKs, indicating that interactions between the CHK active site and the local determinants near the C-terminal regulatory tyrosine of SFKs play only a minor role in governing specific phosphorylation of SFKs by CHK. Our results imply that recognition of SFKs by CHK is mainly governed by interactions between motifs located distally from the active site of CHK and determinants spatially separate from the C-terminal regulatory tyrosine in SFKs. Thus, besides assisting in the identification of potential CHK physiological substrates, our findings shed new light on how CHK recognizes SFKs and other protein substrates.

Amylase production by Preussia minima, a fungus of endophytic origin: optimization of fermentation conditions and analysis of fungal secretome by LC-MS

“…After reduction and alkylation, gel pieces from each protein band were washed twice with 200 μL of a 1:1 (v/v) 100 mM NH 4 HCO 3 /acetonitrile mixture for 15 min, followed by dehydration with 200 μL of 100% acetonitrile, and air-dried for 5 min. Dry gel pieces were rehydrated with 40 μL of 50 mM NH 4 HCO 3 containing 250 ng of trypsin (Sigma) and then incubated at 37°C overnight [ 17 ].…”

β-Synuclein In the Pathogenesis of Parkinson‘s Disease and Related α-Synucleinopathies: Emerging Roles and New Directions

“…It is reasonable to hypothesize that phosphorylation of these tyrosine residues could affect the propensity of bS to aggregate, since aS aggregation is negatively regulated by phosphorylation of the corresponding tyrosine residues (i.e., Y125, Y133 and Y136) in aS [44,45]. Indeed, it was recently shown that Y127 in bS is preferentially phosphorylated by the C-terminal Src kinase-homologous kinase (CHK) [46]. Furthermore, it should be noted that bS is a major o-glycosylated protein in the presynaptic regions of the rodent brain, together with UCH-L1 (PARK5) and collapsin response mediator protein-2 [47].…”

Global Analysis of Human Nonreceptor Tyrosine Kinase Specificity Using High-Density Peptide Microarrays

“…In addition, peptides that are derived from authentic protein substrates of kinases can lack key determinants of specificity and thus make inefficient substrates, as in the case of the phosphorylation of the C-terminal tail of Src family kinases by their upstream regulators CSK and CHK. 21 …”