Granzyme K Displays Highly Restricted Substrate Specificity That Only Partially Overlaps with Granzyme A

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 β-tubulin after two distinct Arg residues. Neither GrK cleavage sites in β-tubulin nor a peptide-based 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. 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 β-tubulin after two distinct Arg residues. Neither GrK cleavage sites in β-tubulin nor a peptide-based 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 (1Lieberman J. Nat. Rev. Immunol.. 2003; 3: 361-370Google Scholar, 2Chowdhury D. Lieberman J. Annu. Rev. Immunol.. 2008; 26: 389-420Google Scholar). 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) 2The abbreviations used are: Gr, granzyme; 2D-DIGE, fluorescent two-dimensional difference gel electrophoresis; hnRNP K, heterogeneous nuclear ribonucleoprotein K; MS, mass spectrometry; SA, serine to alanine mutation; Chaps, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid. 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 single-stranded DNA damage, apoptotic morphology, mitochondrial dysfunction, and loss of cell membrane integrity and occurs independent of caspases and the GrB-induced apoptotic routes (1Lieberman J. Nat. Rev. Immunol.. 2003; 3: 361-370Google Scholar, 2Chowdhury D. Lieberman J. Annu. Rev. Immunol.. 2008; 26: 389-420Google Scholar, 3Martinvalet D. Zhu P. Lieberman J. Immunity.. 2005; 22: 355-370Google Scholar, 4Fan Z. Beresford P.J. Oh D.Y. Zhang D. Lieberman J. Cell.. 2003; 112: 659-672Google Scholar, 5Pardo J. Bosque A. Brehm R. Wallich R. Naval J. Müllbacher A. Anel A. Simon M.M. J. Cell Biol.. 2004; 167: 457-768Google Scholar). GrA is targeted inside the mitochondrion (6Martinvalet D. Dykxhoorn D.M. Ferrini R. Lieberman J. Cell.. 2008; 133: 681-692Google Scholar), where it triggers an increase in reactive oxygen species and loss of transmembrane potential (3Martinvalet D. Zhu P. Lieberman J. Immunity.. 2005; 22: 355-370Google Scholar, 5Pardo J. Bosque A. Brehm R. Wallich R. Naval J. Müllbacher A. Anel A. Simon M.M. J. Cell Biol.. 2004; 167: 457-768Google Scholar). 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 (4Fan Z. Beresford P.J. Oh D.Y. Zhang D. Lieberman J. Cell.. 2003; 112: 659-672Google Scholar), DNA-binding protein HMG-2 (7Fan Z. Beresford P.J. Zhang D. Lieberman J. Mol. Cell. Biol.. 2002; 22: 2810-2820Google Scholar), and base excision repair enzyme Ape1 (8Fan Z. Beresford P.J. Zhang D. Xu Z. Novina C.D. Yoshida A. Pommier Y. Lieberman J. Nat. Immunol.. 2003; 4: 145-153Google Scholar). Cleavage of SET by GrA allows the SET complex component DNase NM23H1 to make single-stranded nicks in the chromosomal DNA. GrA also facilitates DNA damage by cleavage of Ape1 and the double-stranded DNA repair protein Ku70 (9Zhu P. Zhang D. Chowdhury D. Martinvalet D. Keefe D. Shi L. Lieberman J. EMBO Rep.. 2006; 7: 431-437Google Scholar). GrA is a highly specific serine protease in that only ∼10 substrates have been identified and verified as physiological substrates within cells (1Lieberman J. Nat. Rev. Immunol.. 2003; 3: 361-370Google Scholar, 2Chowdhury D. Lieberman J. Annu. Rev. Immunol.. 2008; 26: 389-420Google Scholar). Far less is known about the molecular mechanism and function of GrK. GrK is similar to GrA in that they are closely linked on the same chromosome and that they are the only granzymes that display tryptase-like activity, i.e. both granzymes cleave after basic residues Arg and Lys (10Mahrus S. Craik C.S. Chem. Biol.. 2005; 12: 567-577Google Scholar). This tryptase-like activity contributes to cytotoxic lymphocyte-induced target cell death (11Carter C.R. Sayers T.J. Wiltrout R.H. Turcovski-Corrales S.M. Taub D.D. Cell. Immunol.. 1996; 172: 235-245Google Scholar). GrA-deficient mice or cytotoxic lymphocytes thereof have normal expression levels of GrK and display almost normal cytolytic activity against tumor targets (12Shresta S. Goda P. Wesselschmidt R. Ley T.J. J. Biol. Chem.. 1997; 272: 20236-20244Google Scholar, 13Davis J.E. Smyth M.J. Trapani J.A. Eur. J. Immunol.. 2001; 31: 39-47Google Scholar). Therefore, it is believed that GrK provides a backup and failsafe mechanism for GrA with redundant specificity. Indeed, recent investigations have indicated that GrK induces cell death with similar hallmarks as GrA. GrK triggers rapid caspase-independent cell death with nuclear morphological changes, single-stranded DNA nicks, and reactive oxygen species production from mitochondria (3Martinvalet D. Zhu P. Lieberman J. Immunity.. 2005; 22: 355-370Google Scholar, 14Zhao T. Zhang H. Guo Y. Zhang Q. Hua G. Lu H. Hou Q. Liu H. Fan Z. Cell Death Differ.. 2007; 14: 489-499Google Scholar, 15MacDonald G. Shi L. Vande Velde C. Lieberman J. Greenberg A.H. J. Exp. Med.. 1999; 189: 131-144Google Scholar). Like GrA, GrK efficiently cleaves the SET complex components ApeI, HMG2, and SET, allowing the DNase NM23H1 to make single-stranded nicks in the chromosomal DNA (7Fan Z. Beresford P.J. Zhang D. Lieberman J. Mol. Cell. Biol.. 2002; 22: 2810-2820Google Scholar, 8Fan Z. Beresford P.J. Zhang D. Xu Z. Novina C.D. Yoshida A. Pommier Y. Lieberman J. Nat. Immunol.. 2003; 4: 145-153Google Scholar, 14Zhao T. Zhang H. Guo Y. Zhang Q. Hua G. Lu H. Hou Q. Liu H. Fan Z. Cell Death Differ.. 2007; 14: 489-499Google Scholar, 16Beresford P.J. Zhang D. Oh D.Y. Fan Z. Greer E.L. Russo M.L. Jaju M. Lieberman J. J. Biol. Chem.. 2001; 276: 43285-43293Google Scholar). GrK cleaves SET, ApeI, and HMG2 with similar degradation fragments as GrA, strongly suggesting that GrK and GrA cleave these proteins at the same sites (14Zhao T. Zhang H. Guo Y. Zhang Q. Hua G. Lu H. Hou Q. Liu H. Fan Z. Cell Death Differ.. 2007; 14: 489-499Google Scholar). These findings further strengthen the contention that GrK and GrA induce similar cell death mechanisms with redundant specificity and function. In the present study, we addressed the question of whether GrK indeed displays identical substrate specificity as GrA. We employed peptide- and protease-proteomic screens to show that GrK and GrA display highly restricted substrate specificities that overlap only partially. Whereas three SET complex components are cleaved by both GrA and GrK likely at the same sites (14Zhao T. Zhang H. Guo Y. Zhang Q. Hua G. Lu H. Hou Q. Liu H. Fan Z. Cell Death Differ.. 2007; 14: 489-499Google Scholar), we show that both granzymes also cleave the pre-mRNA-binding protein heterogeneous ribonuclear protein K (hnRNP K) with different kinetics at different sites. Furthermore, we demonstrate that GrK, but not GrA, cleaves the microtubule network protein β-tubulin after two distinct Arg residues. These data suggest 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. Cell Lines, Antibodies, and Reagents—Jurkat cells in with and protein from cells. The cells two in a containing and and in the same by three of The for at at and protein stored at used K and hnRNP K by A. protein was on a protein was by the of GrA and GrK from and and the expression GrA and GrK, in in the is by was by The the of and granzymes in for as by the GrA, GrK, and and to by as for P.J. R. R. M. J.A. J. Immunol.. 2008; Scholar). against and and stored at Both GrA and GrK they the substrates and not and β-tubulin from and the expression and K proteins in as by the by protein was by against and stored at to cells in and to three of and the by at for with GrA GrK or After at the the two-dimensional as by the and in Chaps, and and of a of with of or of both with functions as The by with Chaps, and and to the The for at to in the for for in and for in the same containing of The to a gel to a fluorescent and for at The and on a at the and was at and a was to proteins from the The of gel was by and the was used as A. L. C. S. 2003; 3: Scholar). of gel was by was as by mass and of was as T. J. M. T. A. P.J. L. J. Exp. Med.. 2006; The to and on a on an at a of with a from to The was to a mass was from mass and in at a of The data and to data base or against and the data with a mass for both and The identified by of the used to the substrate of GrK and GrA. and at the a allowing cleavage between the and The to the the and in on The containing GrA GrK and in and or on with a for at in a the three for at in and with in at for by three for at in and After with the and The and the The between granzyme and from at The from that at two GrK and GrA SET with have GrK, GrA, and thereof as and that GrA the GrK as a S. Craik C.S. Nat. Biol.. 2003; Scholar, C. Nat. Biol.. 2003; Scholar, C. P. J. Biol. Chem.. 2002; Scholar). Both granzymes they Arg and Lys substrates not (10Mahrus S. Craik C.S. Chem. Biol.. 2005; 12: 567-577Google Scholar). In with the (9Zhu P. Zhang D. Chowdhury D. Martinvalet D. Keefe D. Shi L. Lieberman J. EMBO Rep.. 2006; 7: 431-437Google Scholar, 14Zhao T. Zhang H. Guo Y. Zhang Q. Hua G. Lu H. Hou Q. Liu H. Fan Z. Cell Death Differ.. 2007; 14: 489-499Google Scholar, 16Beresford P.J. Zhang D. Oh D.Y. Fan Z. Greer E.L. Russo M.L. Jaju M. Lieberman J. J. Biol. Chem.. 2001; 276: 43285-43293Google Scholar), of tumor cell with GrK or GrA in cleavage of SET, was by the of the SET protein and the of a cleavage Both cleavage strongly suggesting that GrK and GrA cleave SET at the same (9Zhu P. Zhang D. Chowdhury D. Martinvalet D. Keefe D. Shi L. Lieberman J. EMBO Rep.. 2006; 7: 431-437Google Scholar, 14Zhao T. Zhang H. Guo Y. Zhang Q. Hua G. Lu H. Hou Q. Liu H. Fan Z. Cell Death Differ.. 2007; 14: 489-499Google Scholar, 16Beresford P.J. Zhang D. Oh D.Y. Fan Z. Greer E.L. Russo M.L. Jaju M. Lieberman J. J. Biol. Chem.. 2001; 276: 43285-43293Google Scholar). These data that GrK and GrA are and cleave SET with similar GrK and GrA used in with to the substrate specificities of GrK and GrA This protease-proteomic screen the of tumor cells for substrates of the granzymes. tumor with GrK, GrA, or their and with and Both and on the same two-dimensional of GrK and GrA of all with was as allowing between The at and specific for fluorescent and the present in in the and granzyme substrates, present in in the the of potential cleavage proteins in proteomic GrK, and the of tumor cell with GrK GrA, and the of tumor cell with GrA The of for GrK and GrA that substrate specificities of both granzymes are highly restricted and that GrK an more restricted substrate specificity as with GrA. proteins cleaved and by both that the substrate specificities of both granzymes overlap in the cleavage that granzyme are by both strongly suggesting that GrA and GrK cleave these proteins at the same P1 cleavage sites. and we to protein from two-dimensional The of potential GrA and GrK substrates are in The substrates that are by both granzymes are and hnRNP unique substrate of GrK is and a unique substrate of GrA is that granzyme not likely the protein levels these data that GrK and GrA display restricted substrate specificities that overlap only of and GrA-induced cleavage identified by and with GrK nuclear ribonucleoprotein nuclear ribonucleoprotein GrA nuclear ribonucleoprotein in a GrK and GrA hnRNP K with at protein that was cleaved by both GrK and GrA is the pre-mRNA-binding protein hnRNP K, a of the hnRNP of proteins that with DNA and their K hnRNP K is for cell and is in expression at and J. 2004; 26: Scholar). gel and from and of the identified as hnRNP GrK cleaved hnRNP K, GrA only cleaved hnRNP K the suggesting This was by both granzymes with tumor cell and hnRNP K cleavage kinetics by Indeed, GrK was markedly more efficient in cleaving hnRNP K as with GrA hnRNP K cleavage the with GrK and GrA that both granzymes cleave hnRNP K at distinct sites. The hnRNP K cleavage by GrK was also by 2D-DIGE, of hnRNP K and the K used in is against residues and of the cleavage revealed in the of hnRNP K the cleavage likely the of hnRNP whether hnRNP K is a granzyme than a substrate of a protease present in tumor cells that is by GrK, we and in both granzymes cleaved K, and for GrK the cleavage was of hnRNP K expression by we to granzyme cleavage sites by These data that GrK and GrA cleave hnRNP K as a substrate with different kinetics at distinct sites. The a of protein that was cleaved by GrK, but not by GrA, and from proteomic screen is the microtubule network component This protein the integrity of the microtubule network and an in cell we and have that and GrB target the of the i.e. P.J. R. R. M. J.A. J. Immunol.. 2008; Scholar, C. P.J. G. P. J. Biol. Chem.. 2006; Scholar, T. J. Cell 2006; Scholar). and from and of the identified as GrK, but not GrA, cleaved β-tubulin after of suggesting that β-tubulin is a and a β-tubulin cleavage in screen GrK of tumor cell This was by both granzymes with tumor cell and β-tubulin cleavage was by Indeed, β-tubulin was only cleaved by GrK a β-tubulin cleavage was by the of β-tubulin the with of the These that GrK, but not GrA, cleaves the microtubule network component β-tubulin in tumor cell GrK after and whether β-tubulin is a specific and GrK than a substrate of a protease present in tumor granzymes or their with Whereas and of β-tubulin with of GrK in the of the protein and the of cleavage of and and The and β-tubulin cleavage fragments also and and the β-tubulin cleavage was by This cleavage at of GrK and that GrK efficiently cleaves these GrA nor GrK cleaved the microtubule and is a substrate of P.J. R. R. M. J.A. J. Immunol.. 2008; and GrB C. P.J. G. P. J. Biol. Chem.. 2006; Scholar, T. J. Cell 2006; Scholar). of the β-tubulin cleavage fragments indicated that GrK cleaves β-tubulin at two sites, i.e. and These potential cleavage sites by three β-tubulin in or a thereof was Although with different cleavage both β-tubulin cleaved by GrK with similar suggesting that both sites are In the was for GrK that GrK cleaves β-tubulin at two sites, i.e. in the sequence and in the sequence Cleavage by GrK after and the two functional of β-tubulin and these cleavage sites are in all β-tubulin from and the around and not show or a consensus sequence that is by both GrK cleavage cleaves β-tubulin after and and and with or GrK for at The proteins by and with and cleavage are of β-tubulin the GrK cleavage sites. by and β-tubulin cleavage fragments and are indicated and to sequence of and of β-tubulin is indicated in that the GrK cleavage sites are in GrK and GrA P1 Arg and been that both GrK and GrA cleave after an Arg or Lys at the P1 for in substrates is with after (10Mahrus S. Craik C.S. Chem. Biol.. 2005; 12: 567-577Google Scholar). of specificities for both GrK and GrA but and clear consensus sequence been identified (10Mahrus S. Craik C.S. Chem. Biol.. 2005; 12: 567-577Google This protease sequence that are to and to the In the present study, we have a to the substrate specificity of proteases that the potential to sites and sites that We have employed a of to the substrate specificities of GrK and GrA. The was with GrK, GrA, or their that cleaved by GrK or GrA are in Both GrK and GrA cleaved that are in Arg but not Lys residues a for Arg Lys at the P1 for both granzymes. GrK cleaved and GrA cleaved and was cleaved by GrA at the GrA cleaved that a basic cleavage of was by both that GrK and GrA display but also unique substrate specificities This is with the from screen of that a Arg or Lys residue, clear consensus sequence or of the P1 identified for both granzymes GrA, is a for a at and residues at the and GrK, is a for residues at and residues to at and The of a consensus sequence is with known cleavage sites in substrates of GrA (7Fan Z. Beresford P.J. Zhang D. Lieberman J. Mol. Cell. Biol.. 2002; 22: 2810-2820Google Scholar, S. Craik C.S. Nat. Biol.. 2003; or GrK suggesting that specificity from These data that both GrK and GrA P1 Arg and display substrate with clear at the cleavage of that are cleaved by GrK or GrA by in a all the GrK and GrA are the only two This activity is for cytotoxic lymphocytes to their target cells (11Carter C.R. Sayers T.J. Wiltrout R.H. Turcovski-Corrales S.M. Taub D.D. Cell. Immunol.. 1996; 172: 235-245Google Scholar). GrA-deficient cytotoxic cells activity and normal levels of GrK, and their cytolytic activity is (12Shresta S. Goda P. Wesselschmidt R. Ley T.J. J. Biol. Chem.. 1997; 272: 20236-20244Google Scholar, 13Davis J.E. Smyth M.J. Trapani J.A. Eur. J. Immunol.. 2001; 31: 39-47Google Scholar). Therefore, GrK a likely to or with GrA. the been that GrK indeed GrA Like GrA, GrK efficiently induces caspase-independent cell death, characterized by mitochondrial and DNA Although the of mitochondrial damage D. Lieberman J. Annu. Rev. Immunol.. 2008; 26: 389-420Google Scholar, 14Zhao T. Zhang H. Guo Y. Zhang Q. Hua G. Lu H. Hou Q. Liu H. Fan Z. Cell Death Differ.. 2007; 14: 489-499Google Scholar, 15MacDonald G. Shi L. Vande Velde C. Lieberman J. Greenberg A.H. J. Exp. Med.. 1999; 189: 131-144Google Scholar, T. Zhang H. Guo Y. Fan Z. J. Biol. Chem.. 2007; Scholar), GrK induces nuclear nuclear and single-stranded DNA by of three SET complex that are also cleaved by GrA D. Lieberman J. Annu. Rev. Immunol.. 2008; 26: 389-420Google Scholar, 14Zhao T. Zhang H. Guo Y. Zhang Q. Hua G. Lu H. Hou Q. Liu H. Fan Z. Cell Death Differ.. 2007; 14: 489-499Google Scholar). In study, we addressed the question of whether GrK indeed displays identical substrate specificity as GrA. We employed and proteomic screens to show that GrK and GrA display highly restricted substrate specificities that overlap only in and Whereas three SET complex components are cleaved by both GrA and GrK likely at the same sites (14Zhao T. Zhang H. Guo Y. Zhang Q. Hua G. Lu H. Hou Q. Liu H. Fan Z. Cell Death Differ.. 2007; 14: 489-499Google we that both granzymes also cleave the pre-mRNA-binding protein hnRNP K with different kinetics at different sites Furthermore, we demonstrate that GrK, but not GrA, cleaves the microtubule network protein β-tubulin after two distinct Arg residues their activity, both granzymes have their own substrate specificity. These data to hypothesize that GrK not only constitutes a redundant functional backup mechanism that assists GrA-induced cell death, but it also displays a unique function by cleaving its own specific substrates. The proteomic revealed only a set of potential GrK and GrA substrates and The of cleavage in a tumor cell that GrK and GrA display restricted substrate specificity. GrB a substrate to cleavage and is with an GrA and GrK cleave substrates not D. Scholar, D. J. Chem.. Scholar). This that the substrate specificities of GrA and GrK are not restricted to the and on sites to the that with residues around P1 in the substrate interactions between exosites of the substrate and the enzyme. around the P1 to a in granzyme that GrA and GrK cleave a restricted of with sequence in Indeed, of the an Arg or Lys residue, both granzymes cleaved only of the with Although only a of all we not a consensus around the P1 cleavage sites in these and Craik (10Mahrus S. Craik C.S. Chem. Biol.. 2005; 12: 567-577Google used of to show that specificities of both GrK and GrA but and GrB and clear consensus sequence In with GrA and GrK cleave substrates only than or substrates D. Scholar, D. J. Chem.. Scholar). The of a clear consensus around the P1 further is with known cleavage sites in substrates of GrA (7Fan Z. Beresford P.J. Zhang D. Lieberman J. Mol. Cell. Biol.. 2002; 22: 2810-2820Google Scholar, S. Craik C.S. Nat. Biol.. 2003; or GrK These strongly suggest that substrate specificity of GrK and GrA from i.e. interactions between granzyme and The GrK is highly with on its C. P. J. Biol. Chem.. 2002; Scholar). These provide sites for and of substrates to the This is by that the is with the activity of GrK a Lys substrate is only by the of and J. A. also been for the electrostatic between GrA and its substrate SET C. Nat. Biol.. 2003; Scholar). Therefore, in substrate specificity between GrK and GrA at in by in the sequence of In it that GrK is a C. P. J. Biol. Chem.. 2002; Scholar), the of GrA as a S. Craik C.S. Nat. Biol.. 2003; Scholar, C. Nat. Biol.. 2003; GrA is believed to further to GrA specificity in a unique by the S. Craik C.S. Nat. Biol.. 2003; Scholar, C. Nat. Biol.. 2003; Scholar), a that is not by GrK. We have identified potential substrates of both GrK and GrA by proteomic screen not of the known GrK or GrA substrates, of the SET complex (1Lieberman J. Nat. Rev. Immunol.. 2003; 3: 361-370Google Scholar, 2Chowdhury D. Lieberman J. Annu. Rev. Immunol.. 2008; 26: 389-420Google Scholar). Although two-dimensional gel is of protein on a not all proteins of of molecular or or of gel in Furthermore, proteins are not at In of the known granzyme substrates may have been on the but not identified by the of as a to substrate of different The physiological of β-tubulin and hnRNP K cleavage identified in is not Both proteins in that make for cell is a component of the microtubule network that is for cell of cell cell and intracellular of and L. Nat. Rev. 2004; 4: Scholar). of by or the of the microtubule network by potential and in the death of tumor cells C. P.J. G. P. J. Biol. Chem.. 2006; Scholar, L. Nat. Rev. 2004; 4: Scholar). with and to the microtubule network L. Nat. Rev. 2004; 4: Scholar). We and have that and GrB cleave in that likely contributes to tumor cell death P.J. R. R. M. J.A. J. Immunol.. 2008; Scholar, C. P.J. G. P. J. Biol. Chem.. 2006; Scholar, T. J. Cell 2006; Scholar). GrK not cleave we that GrK efficiently cleaved the β-tubulin of the at two distinct sites and This the that cleavage of β-tubulin by GrK a cell death pathway of granzyme that is different from GrA. granzymes target the different components of microtubule and may a critical cytotoxic lymphocyte-induced death of tumor cells. of hnRNP K in cell death, suggesting that it is an protein for cell A. R. S. 2007; Scholar). hnRNP K by specific J. 2004; 26: Scholar, A. M. M. Cell.. 1997; its cleavage or the of proteins in of the Indeed, C.S. S. L. A. M. J. P. R. S. Mol. Cell. Biol.. 2008; that of hnRNP K a of and of apoptotic proteins apoptotic and the of C.S. S. L. A. M. J. P. R. S. Mol. Cell. Biol.. 2008; Scholar). or not cleavage of hnRNP K by GrA GrK contributes to tumor cell death an question that further to their in cell it been that cell hnRNP K and are for and M. Med.. 2005; Scholar, 2005; Scholar). Granzymes a in the of virus-infected cells in (1Lieberman J. Nat. Rev. Immunol.. 2003; 3: 361-370Google Scholar, 2Chowdhury D. Lieberman J. Annu. Rev. Immunol.. 2008; 26: 389-420Google Scholar). This the that of hnRNP K microtubule function production in cells natural killer cell We R. for