Calpain is a cytosolic "modulator protease" that modulates cellular functions in response to Ca2+. To identify in vivo substrates of calpain, yeast two-hybrid screening was done using the 5-EF-hand (penta-EF-hand; PEF) domain of the micro-calpain large subunit (domain IV), since several possible in vivo substrates for calpain have been previously reported to bind to the 5-EF-hand domains. Other than the regulatory subunit of calpain, which binds to the domain IV, heterogeneous nuclear ribonucleoproteins (hnRNP) K and R were identified, and shown to be proteolyzed by micro-calpain in vitro. When expressed in COS7 cells, hnRNP K and micro-calpain co-localized in the cytosol, and Ca2+-ionophore stimulation of the cells resulted in proteolysis of hnRNP K, indicating that hnRNP K is an in vivo substrate for calpain. Now, hnRNP K is considered to function as a scaffold protein for its binding proteins, such as PKCdelta and C/EBPbeta, which were reported to be calpain substrates, suggesting that hnRNP-K is a scaffold for calpain to proteolyze these proteins.