Background: Abnormal regulation of calpains and Src contributes to stroke-induced brain damage. Results:The abnormally activated calpains cleave Src to generate a truncated Src fragment capable of directing neurons to undergo cell death. Conclusion:A new function of Src in neuronal death is discovered. Significance: Prevention of calpain-mediated cleavage of Src is a potential therapeutic strategy to minimize stroke-induced brain damage.

Excitotoxicity, a major cause of neuronal death in acute and chronic neurodegenerative diseases and conditions such as stroke and Parkinson's disease, is initiated by overstimulation of glutamate receptors, leading to calcium overload in affected neurons. The sustained high concentration of intracellular calcium constitutively activates a host of enzymes, notably the calcium-activated proteases calpains, neuronal nitric oxide synthase (nNOS) and NADPH oxidase (NOX), to antagonise the cell survival signalling pathways and induce cell death. Upon overactivation by calcium, calpains catalyse limited proteolysis of specific cellular proteins to modulate their functions; nNOS produces excessive amounts of nitric oxide (NO), which, in turn, covalently modifies specific enzymes by S-nitrosylation; and NOX produces excessive amounts of reactive oxygen species (ROS) to inflict damage to key metabolic enzymes. Presumably, key regulatory enzymes governing cell survival and cell death are aberrantly modified and regulated by calpains, NO and ROS in affected neurons; these aberrantly modified enzymes then cooperate to induce the death of affected neurons. c-Src, an Src family kinase (SFK) member, is one of the aberrantly regulated enzymes involved in excitotoxic neuronal death. Herein we review how SFKs are functionally linked to the glutamate receptors and the biochemical and structural basis of the aberrant regulation of SFKs. Results in the literature suggest that SFKs are aberrantly activated by calpain-mediated truncation and S-nitrosylation. Thus, the aberrantly activated SFKs are targets for therapeutic intervention to reduce the extent of brain damage caused by stroke.

Src is critical to both neuronal survival and death. Intact Src sustains neuronal survival. However, in the excitotoxic condition, calpain cleavage of Src generates a neurotoxic truncated Src fragment. Both intact Src and the neurotoxic truncated Src fragment exert their biological actions by controlling the activities of neuroprotective protein kinases.

Excitotoxicity is a neuronal death process initiated by over-stimulation of ionotropic glutamate receptors. Although dysregulation of proteolysis and protein phosphorylation signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remains unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. Our investigation led to the discovery of proteins that, upon proteolysis by calpains, perturb synaptic organization and function. These included key synaptic regulatory proteins including CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ), which we found to undergo calpain-catalyzed proteolytic processing to generate stable truncated fragments with altered activities. We further show that blockade of proteolysis of Src by calpains could protect against neuronal loss in a rat model of neurotoxicity, and that CaMKIIβ and its isoform CaMKIIα undergo differential processing by calpains in mouse brains under physiological conditions and during ischemic stroke. Our findings thus reveal new insights into excitotoxic neuronal death mechanisms and suggest potential therapeutic targets for neurological disorders.

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.