Granzymes are serine proteases stored in cytolytic granules of cytotoxic lymphocytes that eliminate virus-infected and tumor cells. Little is known about the molecular mechanism and function of granzyme (Gr)K. GrK is similar to GrA in that they are the only granzymes that display tryptase-like activity. Both granzymes induce cell death by single-stranded nicking of the chromosomal DNA by cleaving the same components of the endoplasmic reticulum-associated SET complex. Therefore, GrK may provide a backup and failsafe mechanism for GrA with redundant specificity. In the present study, we addressed the question of whether GrK displays identical substrate specificity as GrA. In peptide-and protease-proteomic screens, GrK and GrA displayed highly restricted substrate specificities that overlapped only partially. Whereas GrK and GrA cleave SET with similar efficiencies likely at the same sites, both granzymes cleaved the pre-mRNA-binding protein heterogeneous ribonuclear protein K with different kinetics at distinct sites. GrK was markedly more efficient in cleaving heterogeneous ribonuclear protein K than GrA. GrK, but not GrA, cleaved the microtubule network protein -tubulin after two distinct Arg residues. Neither GrK cleavage sites in -tubulin nor a peptidebased proteomic screen revealed a clear GrK consensus sequence around the P1 residue, suggesting that GrK specificity depends on electrostatic interactions between exosites of the substrate and the enzyme. We hypothesize that GrK not only constitutes a redundant functional backup mechanism that assists GrA-induced cell death but that it also displays a unique function by cleaving its own specific substrates.Important players in the immune defense against tumor cells and virus-infected cells are cytotoxic T lymphocytes and natural killer cells (1, 2). These immune cells predominantly destroy their target cells by releasing the content of their cytolytic granules, containing the pore-forming protein perforin and a set of serine proteases known as granzymes. In humans, five different granzymes (GrA, GrB, GrH, GrK, and GrM) 2 have been identified that all induce cell death by cleaving critical intracellular substrates. Although GrA and GrB have been extensively studied, little is known about the functions and mechanisms of the other granzymes.The GrA cell death pathway is characterized by singlestranded DNA damage, apoptotic morphology, mitochondrial dysfunction, and loss of cell membrane integrity and occurs independent of caspases and the GrB-induced apoptotic routes (1-5). GrA is targeted inside the mitochondrion (6), where it triggers an increase in reactive oxygen species and loss of transmembrane potential (3, 5). After mitochondrial damage, GrA targets a 270 -440-kDa endoplasmic reticulum-associated complex (SET complex) that contains three GrA substrates, i.e. nucleosome assembly protein SET (4), DNA-binding protein HMG-2 (7), and base excision repair enzyme Ape1 (8). Cleavage of SET by GrA allows the SET complex component DNase NM23H1 to make single-stran...