Identification of a nuclear targeting domain in the insertion between helices C and D in protease inhibitor-10

Protease inhibitor 10 (PI-10), an intracellular ovalbumin-serpin, contains a series of basic amino acids in the loop between helices C and D that exhi bit homology to known nuclear targeting signals. Transfection of HeLa cells with plasmids encoding enhanced green fluorescent protein (EGFP) coupled t o PI-10 revealed an intense fluorescence of the nucleus. Immunoblotting dem onstrated a single M-r 80,000 EGFP . PI-10 complex in isolated nuclei. Muta tion of four basic amino acids in the interhelical loop to alanines (i.e. K 74A, K75A, R76A, K77A) resulted in the fluorescent complex being confined t o the cytoplasm, Further evidence for a nuclear targeting signal in this re gion was provided by localization of the fluorescent label to the nucleus i n cells transfected with a plasmid encoding EGFP fused to the 25 amino acid s comprising the interhelical loop of PI-10 (i.e. Arg-63 to Glu-87), wherea s a cytoplasmic distribution was noted for the construct encoding EGFP coup led to the mutated interhelical loop. These data raise the possibility that PI-10 may play a role in regulating protease activity within the nucleus, a property unique in the field of serpin biology.