Fig 1: Determination of the calpain cleavage site of Src in glutamate-treated neurons and in vitro.A, left panel, Western blot of neuronal Src in control neurons, glutamate-treated neurons and glutamate/calpeptin co-treated neurons. ?NSrc: the long C-terminal fragment generated by calpain cleavage. Right panel, fragment ion chromatogram identifying a neo-N-terminal peptide encompassing residues 54 to 77 of neuronal Src (inset) detectable exclusively in neurons treated with glutamate for 30 min. Blue: y ions, Red: b ions. B, schematic diagram depicting the functional domains of intact Src and the calpain cleavage site (red scissors) and formation of a short N-terminal fragment and ?NSrc by calpain cleavage. The cleavage site is mapped to the Unique Lipid Binding Region in the Unique domain. Hence, ?NSrc lacks the ability to bind plasma membrane. C, proteolytic processing of CRMP2 and Src in cortical brain tissue induced by ischemic stroke. Representative Western blot images of Src in lysates of ipsilateral brain cortex of sham-operated mice and mice at the designated time points after ischemic stroke. The same Western blot image of actin was presented in Figure 3F. The abundance ratios of intact Src and truncated Src (?Src) are shown. Number of replicates: n = 4 or 5 for the sham treatment group and the ischemic stroke treatment groups at the designed post-stroke time. The same blot was probed with anti-Src antibody plus anti-mouse Alexa 800 as primary and secondary antibodies. D, left panel, Coomassie blue-stained SDS-PAGE gel of reaction mixtures containing recombinant neuronal Src (R-Src) after incubation with Calpain 1 for 2 min to 120 min in vitro. Boxes with dotted red lines: samples analyzed with the TAILS method. Right panel, the fragment ion chromatogram identifying the deuterated dimethyl-labeled Src (64–77) segment of R-Src as the neo-N-terminal peptide (inset) detected only in the reaction mixture containing R-Src and calpain 1 at 120 min of incubation. Blue: y ions, Red: b ions.
Fig 2: N-terminomic findings unveiled a new mechanism of dysregulation of synaptic neuronal CRMP2 (DPYSL2).A, CRMP2 is cleaved at sites in its C-terminal tail. Inset: the abundance (M/L) ratios of the neo-N-terminal peptides at 30 and 240 min after glutamate treatment. N.D.: not detected. Red scissors: cleavage sites. B, Western blots of lysates from control and glutamate-treated neurons probed with anti-CRMP2 and anti-pT509 CRMP2 antibodies. Loading control: anti-tubulin blot. Blue stars: potential hyper-phosphorylated forms of intact CRMP2 detected by the anti-CRMP2 and anti-pT509 CRMP2 antibodies. C, structure of a phosphomimetic mutant of CRMP2 (PDB accession: 5yz5). The dotted line represents the disordered C-terminal tail region. D, a model depicting the new mechanism of dysregulation of neuronal CRMP2 during excitotoxicity uncovered by our findings. In control neurons, CRMP2 undergoes hierarchical phosphorylation by Cdk5 and GSK3 at sites in the C-terminal tail. Cdk5 phosphorylates the priming site S522. Upon phosphorylation, pS522 binds GSK3, which catalyzes the processive phosphorylation of CRMP2 at three other sites in the order of S518, T514, and T509. In excitotoxic neurons, cleavage of CRMP2 (depicted by scissors) generates a long truncated CRMP2 fragment that lacks the priming site S522, abolishing S522 phosphorylation by Cdk5 and in turn suppressing processive phosphorylation of S518, T514, and T509 by GSK3. The truncation and lack of phosphorylation at T509, T514, and S518 may contribute to the accumulation of the immunoreactive CRMP2 signals at the dendritic blebs shown in panel E. E, fluorescence microscopy images showing actin (phalloidin), CRMP2 and nuclei (DAPI) in control and glutamate-treated neurons. White arrows: dendritic blebs. White dotted rectangles: sections of the images selected to generate the close-up views shown in left bottom panel. Right bottom panel: number of dendritic blebs per mm2 in control and the glutamate treated neurons in three biological replicates. Results are presented as mean ± SD; **p < 0.01, ***p <0.001, one-way ANOVA with Dunnett’s multiple comparison test. F, proteolytic processing of CRMP2 cortical brain tissue induced by an ischemic stroke. Representative Western blot images of CRMP2 in lysates of ipsilateral brain cortex of sham operated mice and mice at the designated time points after ischemic stroke. The same Western blot image of actin was presented in Figure 4C. The abundance ratios of intact CRMP2 and truncated (?CRMP2) are shown. Number of replicates: n = 4 or 5 for the sham treatment group and the ischemic stroke treatment groups at the designed post-stroke time. The blot was probed with anti-CRMP2 plus anti-rabbit Alexa 488 as primary and secondary antibodies and anti-actin plus anti-mouse Alexa 800 as the primary and secondary antibodies.
Supplier Page from Abcam for Anti-CRMP2 (phospho T509) antibody