Figure 4.
Figure 4. Cleavage of human ATP8A1 and predicted calpain cleavage sites in mammalian ATP8A1 orthologs. (A) mRNA expression pattern of the P4-type ATPase and CDC50 genes derived from the genome-wide RNA-seq analysis of human platelet transcriptome.20 Data were extracted from supplementary Table S4 in Rowley et al.20 (B) Protein expression pattern of the P4-type ATPases and CDC50 proteins derived from the quantitative analysis of human platelet protein composition.22 Data were extracted from supplemental Table 3 in Solari et al.22 (C) Washed human platelets (5 × 107) were preincubated with DMSO (DS), CP (50 µg/mL), or QVD (25 µM) at room temperature for 15 minutes, and then treated with vehicle (DMSO) or ABT737 (1 µM; 37°C; 2 hours) in the absence or presence of CaCl2 (2 mM). Platelets were subsequently lysed for western blot analysis. Immunoblots are representative of n = 3 independent experiments. (D) Topology of ATP8A1 and its predicted calpain cleavage site. Transmembrane helices are numbered. Three cytosolic domains involved in the ATPase catalytic cycle are shown as colored circles: nucleotide-binding (N) domain, which binds ATP; phosphorylation (P) domain, which contains the conserved phosphorylation site in the DKTG (Asp-Lys-Thr-Gly) motif; and actuator (A) domain, which has the DGET motif that facilitates the dephosphorylation of the phosphorylated aspartate intermediate.4,66,67 Calpain cleavage sites were predicted using GPS-CCD 1.0 (http://ccd.biocuckoo.org).41 Although several cleavage sites are predicted, the site at position R139 in the A-domain (indicated by a red box) generates a fragment of around 100 kDa, which is consistent with the western blot fragment size in apoptotic platelets. (E) Amino acid sequence alignment around the calpain cleavage site in 6 mammalian ATP8A1 orthologs: human (UniProt, Q9Y2Q0), mouse (P70704), bovine (Q29449), sheep (W5PYS1), dog (F1PHG9), and rabbit (G1TF29). Sequences are aligned. *Fully conserved residues. The calpain recognition sequence is shown in red. A vertical bar (|) indicates the cleavage site.

Cleavage of human ATP8A1 and predicted calpain cleavage sites in mammalian ATP8A1 orthologs. (A) mRNA expression pattern of the P4-type ATPase and CDC50 genes derived from the genome-wide RNA-seq analysis of human platelet transcriptome.20  Data were extracted from supplementary Table S4 in Rowley et al.20  (B) Protein expression pattern of the P4-type ATPases and CDC50 proteins derived from the quantitative analysis of human platelet protein composition.22  Data were extracted from supplemental Table 3 in Solari et al.22  (C) Washed human platelets (5 × 107) were preincubated with DMSO (DS), CP (50 µg/mL), or QVD (25 µM) at room temperature for 15 minutes, and then treated with vehicle (DMSO) or ABT737 (1 µM; 37°C; 2 hours) in the absence or presence of CaCl2 (2 mM). Platelets were subsequently lysed for western blot analysis. Immunoblots are representative of n = 3 independent experiments. (D) Topology of ATP8A1 and its predicted calpain cleavage site. Transmembrane helices are numbered. Three cytosolic domains involved in the ATPase catalytic cycle are shown as colored circles: nucleotide-binding (N) domain, which binds ATP; phosphorylation (P) domain, which contains the conserved phosphorylation site in the DKTG (Asp-Lys-Thr-Gly) motif; and actuator (A) domain, which has the DGET motif that facilitates the dephosphorylation of the phosphorylated aspartate intermediate.4,66,67  Calpain cleavage sites were predicted using GPS-CCD 1.0 (http://ccd.biocuckoo.org).41  Although several cleavage sites are predicted, the site at position R139 in the A-domain (indicated by a red box) generates a fragment of around 100 kDa, which is consistent with the western blot fragment size in apoptotic platelets. (E) Amino acid sequence alignment around the calpain cleavage site in 6 mammalian ATP8A1 orthologs: human (UniProt, Q9Y2Q0), mouse (P70704), bovine (Q29449), sheep (W5PYS1), dog (F1PHG9), and rabbit (G1TF29). Sequences are aligned. *Fully conserved residues. The calpain recognition sequence is shown in red. A vertical bar (|) indicates the cleavage site.

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