Figure 3.

Establishment of the major calpain-recognition sequence in Kidins220 C-terminus. (a) Immunoblot analysis of protein extracts obtained from HEK293T cells expressing Kidins220-GFP-Ct subjected to in vitro digestion with purified calpain I (20 or 80 U/ml) for 15 or 30 min. The GFP antibody reveals processing of FL Kidins220-GFP-Ct (FL-GFP) and the corresponding accumulation of a major C-terminal GFP-fused fragment (Ct-0-GFP) together with low amounts of additional fragments (Ct-1-GFP/Ct-5-GFP). Spectrin processing and levels of β-actin were used as controls of calpain activity and protein loading, respectively. (b) Schematic representation of fusion protein Kidins220-GFP-Ct with putative minor (Ct-1-GFP/Ct-5-GFP) and major (Ct-0-GFP) calpain targets (asterisks), their approximate location being inferred from immunoblots with antibodies specific for GFP. The fragments produced, together with their estimated molecular weights, are indicated by horizontal arrows. The contribution of GFP to the molecular weight of those fragments is depicted separately (green box). (c) Purification of Ct-0-GFP and Edman sequencing. Total lysates (TL) from HEK293T cells transfected with Kidins220-GFP-Ct were left undigested or subjected to a preparative digestion with purified calpain (80 U/ml). Digested lysates were then immunoprecipitated (IP) with GFP antibodies. Small fractions of total lysates or immunoprecipitates were analyzed with GFP antibodies to corroborate the accumulation and concentration of Ct-0-GFP fragment (highlighted by a red box). The band corresponding to this fragment was cut from a preparative filter stained with Coomasie Blue and subjected to Edman degradation. The N-terminal sequence obtained and the amino acids position (1670–1675) in rat protein (NP_446247) are shown below. (d) Comparison of rat Kidins220 region containing the major calpain cleavage site identified above to homologous regions in five additional vertebrate species, human (NP_065789), mice (NP_001074847), chicken (XP_419939), frog (NP_001120159) and zebrafish (AAH61450). A high level of sequence conservation is observed in all vertebrate species analyzed and, particularly, a seven amino acids stretch comprising Kidins220 calpain-processing site 0 (blue) is completely conserved