- Email: [email protected]
Calpain: a role in cell transformation and migration
The International Journal of Biochemistry & Cell Biology 34 (2002) 1539–1543
Calpain: a role in cell transformation and migration Neil O. Carragher a,∗ , Margaret C. Frame b a
b
The Beatson Institute for Cancer Research, Cancer Research UK Beatson Laboratories, Glasgow G61 1BD, UK Institute of Biological and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK Received 13 March 2002; received in revised form 6 May 2002; accepted 6 May 2002
Abstract Calpains represent a well conserved family of calcium-dependent proteolytic enzymes. Recent progress in determining the three-dimensional crystal structure of calpains and generation of calpain knock out animals have significantly advanced our understanding of both the activation mechanism and physiological role of this protease family. Studies applying molecular intervention strategies and genetic ablation of calpain now provide indisputable evidence that calpain activity contributes to remodelling of the actin cytoskeleton, cell migration and oncogenic transformation. Src and epidermal growth factor receptor (EGFR) stimulated cell motility is dependent upon calpain activation. In addition, calpain promotes accelerated cell-cycle progression and anchorage-independent growth of Src transformed cells. In vivo studies demonstrate a link between calpain expression levels and activity with tumour development and invasion. Thus, recent investigations suggest that the role of calpain in promoting cell transformation and cell migration may have important in vivo consequences in the context of cancer pathobiology. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Calpain; Src; EGFR; Transformation; Migration
1. Introduction Calpains were originally identified as a unique class of calcium activated proteolytic enzymes present in the cytosolic fraction of brain extracts [1]. These proteases were later named calpains to reflect their calcium dependency and homology with the protease domain of the papain family of cysteine proteases. Further studies demonstrate that several mammalian isoforms of calpain exist (calpains 1–13) which are widely expressed in most tissues [2]. Calpain activity is tightly regulated by its ubiquitously expressed endogenous inhibitor calpastatin. Calpains result in the ∗ Corresponding author. Tel.: +44-141-330-3956; fax: +44-141-942-6521. E-mail address: [email protected] (N.O. Carragher).
proteolysis of a broad spectrum of cellular proteins and a distinguishing feature of their activity is their ability to confer limited cleavage of protein substrates into stable fragments rather than complete proteolytic digestion. Thus, calpain-mediated proteolysis represents a major pathway of post-translational modification that influences various aspects of cell physiology, including apoptosis, cell migration and cell proliferation [2]. The recent application of improved pharmacological inhibitors in combination with molecular intervention and calpain gene knock out (KO) strategies has led to recent advances in the identification of key calpain substrates and elucidation of the mechanisms by which calpain regulates cell behaviour. These studies demonstrate a role for calpain activity in modulating cell-cycle control, cell adhesion, spreading, migration
1357-2725/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 2 ) 0 0 0 6 9 - 9
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N.O. Carragher, M.C. Frame / The International Journal of Biochemistry & Cell Biology 34 (2002) 1539–1543
Fig. 1. (A) The 80 kDa catalytic subunit of calpains 1 and 2 can be subdivided into four domains: domain I is a short pro-domain region; domain II represents a distinct papain-like cysteine protease domain; domain III has no significant homology to any other protein, structural analysis indicates that domain III physically associates with domain II, leading to speculation that domain III may regulate protease activity; domain IV is a calcium binding domain that has been proposed to play a role in substrate recognition. The small 30 kDa regulatory subunit (calpain 4) consists of two domains, V and VI. Domain IV of the catalytic subunit and domain VI of the regulatory subunit, each possess five sets of EF hand calcium binding motifs that have been proposed to confer calcium dependency upon the activity of calpain. To date at least another 12 mammalian calpain proteins demonstrating homology to the large catalytic subunit have been identified. (B) Analysis of the three-dimensional crystal structure of human calpain 2 (reproduced with permission from Strobl et al. [3]) indicates that under calcium free conditions the catalytic protease domain II is spatially subdivided into two sub-domains, IIa and IIb resulting in disruption of the active site triad Cys105L-His262L-Asn286L. Researchers have speculated that calcium binding may disrupt the electrostatic interaction between sub domain IIb with domain III allowing sub domain IIb to fuse with IIa forming a functional catalytic domain. Thus, the activation of calpain by calcium appears to be dependent on a conformation switch rather than proteolysis of a pro-peptide region that characterises the activation of other cysteine proteases. Colours of the schematic domain structure (A) correspond with those of the crystal structure (B).
N.O. Carragher, M.C. Frame / The International Journal of Biochemistry & Cell Biology 34 (2002) 1539–1543
and oncogene-induced cell transformation. The mechanisms by which calpain activity is regulated by oncogenes and migratory stimuli are beginning to be elucidated.
2. Structure Calpains 1 and 2 are the most well characterised calpain isoforms. These proteins are heterodimeric enzymes composed of a unique 80 kDa catalytic subunit (calpains 1 and 2) associated with a common regulatory subunit (calpain 4). The catalytic subunit confers proteolytic activity and can be subdivided into four functional domains as illustrated in Fig. 1a. The threedimensional crystal structure of calpain 2 in the absence of calcium has recently been described (Fig. 1B) [3]. The calpain inhibitory domains of calpastatin and the protease domain of calpain are highly conserved between species. A variety of atypical catalytic calpain subunit homologues have been described in lower organisms including, drosophila, nematodes, fungi, yeast, and bacteria. This conservation of calpain-like proteases and their inhibitor suggests that the calpaincalpastatin proteolytic system provides distinct and essential functions for many living organisms.
3. Synthesis and degradation The calpain catalytic subunits (calpains 1, 2, 5, 7, 10 and 12) and the small regulatory subunit (calpain 4) are ubiquitously expressed in all tissues. Several calpain homologues (calpains 3, 8, 9, 11, 6 and 13) have been demonstrated to be expressed in a tissue specific manner, while calpastatin is found ubiquitously within all human tissues. Alternative splicing and proteolytic processing can generate a number of tissue specific calpastatin isoforms. Under normal physiological conditions the main calpain enzymes and their inhibitor calpastatin have been reported to exist as relatively stable proteins with a half-life of up to 5 days [4]. However, in response to particular stimuli, such as elevated intracellular calcium, ischemic injury or activation of the oncoprotein v-Src, calpain can result in the proteolysis of its inhibitor calpastatin, thereby enhancing calpain activity by a positive feedback loop [5].
1541
4. Biological function Recent application of molecular intervention strategies targeted against calpain activity, such as overexpression of calpastatin or generation of calpain KO animals and cells, have advanced our understanding of the role calpain plays in cell behaviour and have begun to revolutionise this field of study. Homozygous KO of the calpain 4 gene that encodes the small regulatory subunit of calpains 1 and 2 containing heterodimers, results in a lethal phenotype. Calpain 4 KO embryos die at around 10 days post-conception and exhibit a defect in vascular development suggesting a role for calpain in endothelial cell proliferation, migration or survival. Fibroblasts derived from calpain 4 KO embryos exhibit impaired cell migration. The actin cytoskeleton, formation of filipodia, distribution of the integrin-linked focal adhesion complexes and proteolytic cleavage of the focal adhesion component talin are all altered in calpain 4 KO cells [6]. Integrin-linked focal adhesion complexes provide the main adhesive links between the cellular actin cytoskeleton and the surrounding extracellular matrix. It has been proposed that cells utilise a complex temporal and spatially regulated mechanism of focal adhesion assembly at the leading edge, co-ordinated with focal adhesion disassembly at the cell rear to permit cell migration. Several studies indicate that calpains localise to integrin-associated complexes. Furthermore, many of the protein components of focal adhesions are known substrates of calpain. Calpain cleavage of focal adhesion components, focal adhesion kinase (FAK), paxillin, talin and possibly others promotes the disassembly of these complexes contributing to reduced cell adhesion and increased motility [7,8]. Calpain inhibitor studies indicate that calpain activity is required to specifically release integrin contacts at the cell rear to permit organised cell migration [9]. Recent investigations demonstrate that epidermal growth factor receptor (EGFR) induced ERK/MAPK signalling promotes calpain activity contributing to EGF induced substrate de-adhesion and increased cell motility [10]. We have recently reported that v-Src induced transformation of primary embryo fibroblasts is also accompanied by calpain-mediated proteolytic cleavage of FAK, focal adhesion disassembly and enhanced motility of Src-transformed cells [8]. Calpain activity also contributes to accelerated cell cycle
1542
N.O. Carragher, M.C. Frame / The International Journal of Biochemistry & Cell Biology 34 (2002) 1539–1543
Fig. 2. Activation of v-Src induces protein synthesis of calpain 2 leading to calpain-mediated degradation of its own inhibitor calpastatin thereby, further enhancing calpain activity. Increased calpain activity in v-Src transformed cells promotes proteolytic cleavage of FAK, focal adhesion disassembly, reduced substrate adhesion and increased cell motility [8]. Enhanced calpain activity also accelerates the progression of transformed cells through the G1 stage of the cell-cycle and contributes to anchorage-independent growth [5]. EGFR induced ERK/MAPK activation leads to phosphorylation of calpain and increased calpain activity that subsequently promotes EGF induced substrate de-adhesion and increased motility.
progression and anchorage-independent growth of Src-transformed cells [5] (Fig. 2). Calpain-mediated proteolytic cleavage of FAK has also been detected during cell transformation induced by other oncoproteins such as k-Ras, v-Fos and v-Myc indicating that elevated calpain activity may promote cell transformation induced by other oncoproteins ([5]; Carragher et al., unpublished). Interestingly, studies indicate that the v-FosFBR oncoprotein is more resistant to
calpain-mediated proteolysis compared to its highly susceptible cellular counterpart c-Fos. These investigations indicate that decreased calpain-mediated turnover of v-FosFBR may contribute to the transformation and tumourigenic potential of v-FosFBR infected cells [11]. In contrast to these studies, a recent report suggests that calpain-mediated cleavage of PKCε may suppress transformation of NIH3T3 mouse fibroblasts [12]. Such conflicting roles for calpain in
N.O. Carragher, M.C. Frame / The International Journal of Biochemistry & Cell Biology 34 (2002) 1539–1543
regulating the transformed state of cells are likely a consequence of contrasting substrate specificity determined by cell type and the particular oncoprotein responsible for instigating cell transformation.
5. Possible medical applications Calpain activity has been implicated in numerous pathological conditions including, Alzheimer’s disease, demyelination events of multiple sclerosis, neuronal damage after spinal cord injury and hypoxic/ ischaemic injury to brain, kidney and heart organs. Three separate studies point to the importance of calpain activity during tumour development and invasion. In human renal cell carcinomas significantly higher levels of calpain I expression are found in tumours that metastasised to peripheral lymph nodes relative to tumours that had not metastasised [13]. Elevated calpain activity was detected in breast cancer tissues relative to normal breast tissues [14] and calpain-mediated proteolysis of the tumour suppressor protein neurofibromatosis type 2 (NF2 or merlin) is associated with the development of schwannomas and meningiomas [15]. The recent development of calpain KO animals and crystallisation of calpain 2 shall advance our understanding of the physiological roles of calpain and their mechanism of activation. This information will facilitate the design of improved specific inhibitors of calpain activity that may present useful therapeutic approaches for the treatment of calpain-associated pathological disorders.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
References [1] G. Guroff, A neutral, calcium-activated proteinase from the soluble fraction of rat brain, J. Biol. Chem. 239 (1964) 149–155. [2] H. Sorimachi, S. Ishiura, K. Suzuki, Structure and physiological function of calpains, Biochem. J. 328 (1997) 721–732. [3] S. Strobl, C. Fernandez-Catalan, M. Braun, R. Huber, H. Masumoto, K. Nakagawa, A. Irie, H. Sorimachi, G. Bourenkow, H. Bartunik, K. Suzuki, W. Bode, The crystal structure
[14]
[15]
1543
of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 588–592. W. Zhang, R.D. Lane, R.L. Mellgren, The major calpain isozymes are long-lived proteins. Design of an antisense strategy for calpain depletion in cultured cells, J. Biol. Chem. 271 (1996) 18825–18830. N.O. Carragher, M.A. Westhoff, D. Riley, D.A. Potter, P. Dutt, J.S. Elce, P.A. Greer, M.C. Frame, v-Src-induced modulation of the calpain-calpastatin proteolytic system regulates transformation, Mol. Cell Biol. 22 (2002) 257–269. N. Dourdin, A.K. Bhatt, P. Dutt, P.A. Greer, J.S. Arthur, J.S. Elce, A. Huttenlocher, Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts, J. Biol. Chem. 276 (2001) 48382–48388. N.O. Carragher, B. Levkau, R. Ross, E.W. Raines, Degraded collagen fragments promote rapid disassembly of smooth muscle focal adhesions that correlates with cleavage of pp125 (FAK), paxillin, and talin, J. Cell Biol. 7 (1999) 619–630. N.O. Carragher, V.J. Fincham, D. Riley, M.C. Frame, Cleavage of focal adhesion kinase by different proteases during SRC- regulated transformation, J. Biol. Chem. 276 (2001) 4270–4275. A. Huttenlocher, S.P. Palecek, Q. Lu, W. Zhang, R.L. Mellgren, D.A. Lauffenburger, M.H. Ginsberg, A.F. Horwitz, Regulation of cell migration by the calcium-dependent protease calpain, J. Biol. Chem. 272 (1997) 32719–32722. A. Glading, P. Chang, D.A. Lauffenburger, A. Wells, Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway, J. Biol. Chem. 275 (2000) 2390–2398. A.M. Steff, S. Carillo, M. Pariat, M. Piechaczyk, Decreased susceptibility to calpains of v-FosFBR but not of v-FosFBJ or v-JunASV17 retroviral proteins compared with their cellular counterparts, Biochem. J. 323 (1997) 685–692. T. Hiwasa, N. Mitsuyuki, M. Nakata, S. Ohno, M. Maki, K. Suzuki, M. Takiguchi, Regulation of transformed state by calpastatin via PKCε in NIH3T3 mouse fibroblasts, Biochem. Biophys. Res. Commun. 290 (2002) 510–517. C. Braun, M. Engel, M. Seifert, B. Theisinger, G. Seitz, K.D. Zang, C. Welter, Expression of calpain I messenger RNA in human renal cell carcinoma: correlation with lymph node metastasis and histological type, Int. J. Cancer 84 (1999) 6–9. E. Shiba, J.I. Kambayashi, M. Sakon, T. Kawasaki, T. Kobayashi, H. Koyama, E. Yayoi, Y. Takatsuka, S.I. Takai, Ca2+ -dependent neutral protease (calpain) activity in breast cancer tissue and estrogen receptor status, Breast Cancer 3 (1996) 13–17. Y. Kimura, H. Saya, M. Nakao, Calpain-dependent proteolysis of NF2 protein: involvement in schwannomas and meningiomas, Neuropathology 20 (2000) 153–160.
Calpain: a role in cell transformation and migration Neil O. Carragher a,∗ , Margaret C. Frame b a
b
The Beatson Institute for Cancer Research, Cancer Research UK Beatson Laboratories, Glasgow G61 1BD, UK Institute of Biological and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK Received 13 March 2002; received in revised form 6 May 2002; accepted 6 May 2002
Abstract Calpains represent a well conserved family of calcium-dependent proteolytic enzymes. Recent progress in determining the three-dimensional crystal structure of calpains and generation of calpain knock out animals have significantly advanced our understanding of both the activation mechanism and physiological role of this protease family. Studies applying molecular intervention strategies and genetic ablation of calpain now provide indisputable evidence that calpain activity contributes to remodelling of the actin cytoskeleton, cell migration and oncogenic transformation. Src and epidermal growth factor receptor (EGFR) stimulated cell motility is dependent upon calpain activation. In addition, calpain promotes accelerated cell-cycle progression and anchorage-independent growth of Src transformed cells. In vivo studies demonstrate a link between calpain expression levels and activity with tumour development and invasion. Thus, recent investigations suggest that the role of calpain in promoting cell transformation and cell migration may have important in vivo consequences in the context of cancer pathobiology. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Calpain; Src; EGFR; Transformation; Migration
1. Introduction Calpains were originally identified as a unique class of calcium activated proteolytic enzymes present in the cytosolic fraction of brain extracts [1]. These proteases were later named calpains to reflect their calcium dependency and homology with the protease domain of the papain family of cysteine proteases. Further studies demonstrate that several mammalian isoforms of calpain exist (calpains 1–13) which are widely expressed in most tissues [2]. Calpain activity is tightly regulated by its ubiquitously expressed endogenous inhibitor calpastatin. Calpains result in the ∗ Corresponding author. Tel.: +44-141-330-3956; fax: +44-141-942-6521. E-mail address: [email protected] (N.O. Carragher).
proteolysis of a broad spectrum of cellular proteins and a distinguishing feature of their activity is their ability to confer limited cleavage of protein substrates into stable fragments rather than complete proteolytic digestion. Thus, calpain-mediated proteolysis represents a major pathway of post-translational modification that influences various aspects of cell physiology, including apoptosis, cell migration and cell proliferation [2]. The recent application of improved pharmacological inhibitors in combination with molecular intervention and calpain gene knock out (KO) strategies has led to recent advances in the identification of key calpain substrates and elucidation of the mechanisms by which calpain regulates cell behaviour. These studies demonstrate a role for calpain activity in modulating cell-cycle control, cell adhesion, spreading, migration
1357-2725/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 2 ) 0 0 0 6 9 - 9
1540
N.O. Carragher, M.C. Frame / The International Journal of Biochemistry & Cell Biology 34 (2002) 1539–1543
Fig. 1. (A) The 80 kDa catalytic subunit of calpains 1 and 2 can be subdivided into four domains: domain I is a short pro-domain region; domain II represents a distinct papain-like cysteine protease domain; domain III has no significant homology to any other protein, structural analysis indicates that domain III physically associates with domain II, leading to speculation that domain III may regulate protease activity; domain IV is a calcium binding domain that has been proposed to play a role in substrate recognition. The small 30 kDa regulatory subunit (calpain 4) consists of two domains, V and VI. Domain IV of the catalytic subunit and domain VI of the regulatory subunit, each possess five sets of EF hand calcium binding motifs that have been proposed to confer calcium dependency upon the activity of calpain. To date at least another 12 mammalian calpain proteins demonstrating homology to the large catalytic subunit have been identified. (B) Analysis of the three-dimensional crystal structure of human calpain 2 (reproduced with permission from Strobl et al. [3]) indicates that under calcium free conditions the catalytic protease domain II is spatially subdivided into two sub-domains, IIa and IIb resulting in disruption of the active site triad Cys105L-His262L-Asn286L. Researchers have speculated that calcium binding may disrupt the electrostatic interaction between sub domain IIb with domain III allowing sub domain IIb to fuse with IIa forming a functional catalytic domain. Thus, the activation of calpain by calcium appears to be dependent on a conformation switch rather than proteolysis of a pro-peptide region that characterises the activation of other cysteine proteases. Colours of the schematic domain structure (A) correspond with those of the crystal structure (B).
N.O. Carragher, M.C. Frame / The International Journal of Biochemistry & Cell Biology 34 (2002) 1539–1543
and oncogene-induced cell transformation. The mechanisms by which calpain activity is regulated by oncogenes and migratory stimuli are beginning to be elucidated.
2. Structure Calpains 1 and 2 are the most well characterised calpain isoforms. These proteins are heterodimeric enzymes composed of a unique 80 kDa catalytic subunit (calpains 1 and 2) associated with a common regulatory subunit (calpain 4). The catalytic subunit confers proteolytic activity and can be subdivided into four functional domains as illustrated in Fig. 1a. The threedimensional crystal structure of calpain 2 in the absence of calcium has recently been described (Fig. 1B) [3]. The calpain inhibitory domains of calpastatin and the protease domain of calpain are highly conserved between species. A variety of atypical catalytic calpain subunit homologues have been described in lower organisms including, drosophila, nematodes, fungi, yeast, and bacteria. This conservation of calpain-like proteases and their inhibitor suggests that the calpaincalpastatin proteolytic system provides distinct and essential functions for many living organisms.
3. Synthesis and degradation The calpain catalytic subunits (calpains 1, 2, 5, 7, 10 and 12) and the small regulatory subunit (calpain 4) are ubiquitously expressed in all tissues. Several calpain homologues (calpains 3, 8, 9, 11, 6 and 13) have been demonstrated to be expressed in a tissue specific manner, while calpastatin is found ubiquitously within all human tissues. Alternative splicing and proteolytic processing can generate a number of tissue specific calpastatin isoforms. Under normal physiological conditions the main calpain enzymes and their inhibitor calpastatin have been reported to exist as relatively stable proteins with a half-life of up to 5 days [4]. However, in response to particular stimuli, such as elevated intracellular calcium, ischemic injury or activation of the oncoprotein v-Src, calpain can result in the proteolysis of its inhibitor calpastatin, thereby enhancing calpain activity by a positive feedback loop [5].
1541
4. Biological function Recent application of molecular intervention strategies targeted against calpain activity, such as overexpression of calpastatin or generation of calpain KO animals and cells, have advanced our understanding of the role calpain plays in cell behaviour and have begun to revolutionise this field of study. Homozygous KO of the calpain 4 gene that encodes the small regulatory subunit of calpains 1 and 2 containing heterodimers, results in a lethal phenotype. Calpain 4 KO embryos die at around 10 days post-conception and exhibit a defect in vascular development suggesting a role for calpain in endothelial cell proliferation, migration or survival. Fibroblasts derived from calpain 4 KO embryos exhibit impaired cell migration. The actin cytoskeleton, formation of filipodia, distribution of the integrin-linked focal adhesion complexes and proteolytic cleavage of the focal adhesion component talin are all altered in calpain 4 KO cells [6]. Integrin-linked focal adhesion complexes provide the main adhesive links between the cellular actin cytoskeleton and the surrounding extracellular matrix. It has been proposed that cells utilise a complex temporal and spatially regulated mechanism of focal adhesion assembly at the leading edge, co-ordinated with focal adhesion disassembly at the cell rear to permit cell migration. Several studies indicate that calpains localise to integrin-associated complexes. Furthermore, many of the protein components of focal adhesions are known substrates of calpain. Calpain cleavage of focal adhesion components, focal adhesion kinase (FAK), paxillin, talin and possibly others promotes the disassembly of these complexes contributing to reduced cell adhesion and increased motility [7,8]. Calpain inhibitor studies indicate that calpain activity is required to specifically release integrin contacts at the cell rear to permit organised cell migration [9]. Recent investigations demonstrate that epidermal growth factor receptor (EGFR) induced ERK/MAPK signalling promotes calpain activity contributing to EGF induced substrate de-adhesion and increased cell motility [10]. We have recently reported that v-Src induced transformation of primary embryo fibroblasts is also accompanied by calpain-mediated proteolytic cleavage of FAK, focal adhesion disassembly and enhanced motility of Src-transformed cells [8]. Calpain activity also contributes to accelerated cell cycle
1542
N.O. Carragher, M.C. Frame / The International Journal of Biochemistry & Cell Biology 34 (2002) 1539–1543
Fig. 2. Activation of v-Src induces protein synthesis of calpain 2 leading to calpain-mediated degradation of its own inhibitor calpastatin thereby, further enhancing calpain activity. Increased calpain activity in v-Src transformed cells promotes proteolytic cleavage of FAK, focal adhesion disassembly, reduced substrate adhesion and increased cell motility [8]. Enhanced calpain activity also accelerates the progression of transformed cells through the G1 stage of the cell-cycle and contributes to anchorage-independent growth [5]. EGFR induced ERK/MAPK activation leads to phosphorylation of calpain and increased calpain activity that subsequently promotes EGF induced substrate de-adhesion and increased motility.
progression and anchorage-independent growth of Src-transformed cells [5] (Fig. 2). Calpain-mediated proteolytic cleavage of FAK has also been detected during cell transformation induced by other oncoproteins such as k-Ras, v-Fos and v-Myc indicating that elevated calpain activity may promote cell transformation induced by other oncoproteins ([5]; Carragher et al., unpublished). Interestingly, studies indicate that the v-FosFBR oncoprotein is more resistant to
calpain-mediated proteolysis compared to its highly susceptible cellular counterpart c-Fos. These investigations indicate that decreased calpain-mediated turnover of v-FosFBR may contribute to the transformation and tumourigenic potential of v-FosFBR infected cells [11]. In contrast to these studies, a recent report suggests that calpain-mediated cleavage of PKCε may suppress transformation of NIH3T3 mouse fibroblasts [12]. Such conflicting roles for calpain in
N.O. Carragher, M.C. Frame / The International Journal of Biochemistry & Cell Biology 34 (2002) 1539–1543
regulating the transformed state of cells are likely a consequence of contrasting substrate specificity determined by cell type and the particular oncoprotein responsible for instigating cell transformation.
5. Possible medical applications Calpain activity has been implicated in numerous pathological conditions including, Alzheimer’s disease, demyelination events of multiple sclerosis, neuronal damage after spinal cord injury and hypoxic/ ischaemic injury to brain, kidney and heart organs. Three separate studies point to the importance of calpain activity during tumour development and invasion. In human renal cell carcinomas significantly higher levels of calpain I expression are found in tumours that metastasised to peripheral lymph nodes relative to tumours that had not metastasised [13]. Elevated calpain activity was detected in breast cancer tissues relative to normal breast tissues [14] and calpain-mediated proteolysis of the tumour suppressor protein neurofibromatosis type 2 (NF2 or merlin) is associated with the development of schwannomas and meningiomas [15]. The recent development of calpain KO animals and crystallisation of calpain 2 shall advance our understanding of the physiological roles of calpain and their mechanism of activation. This information will facilitate the design of improved specific inhibitors of calpain activity that may present useful therapeutic approaches for the treatment of calpain-associated pathological disorders.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
References [1] G. Guroff, A neutral, calcium-activated proteinase from the soluble fraction of rat brain, J. Biol. Chem. 239 (1964) 149–155. [2] H. Sorimachi, S. Ishiura, K. Suzuki, Structure and physiological function of calpains, Biochem. J. 328 (1997) 721–732. [3] S. Strobl, C. Fernandez-Catalan, M. Braun, R. Huber, H. Masumoto, K. Nakagawa, A. Irie, H. Sorimachi, G. Bourenkow, H. Bartunik, K. Suzuki, W. Bode, The crystal structure
[14]
[15]
1543
of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 588–592. W. Zhang, R.D. Lane, R.L. Mellgren, The major calpain isozymes are long-lived proteins. Design of an antisense strategy for calpain depletion in cultured cells, J. Biol. Chem. 271 (1996) 18825–18830. N.O. Carragher, M.A. Westhoff, D. Riley, D.A. Potter, P. Dutt, J.S. Elce, P.A. Greer, M.C. Frame, v-Src-induced modulation of the calpain-calpastatin proteolytic system regulates transformation, Mol. Cell Biol. 22 (2002) 257–269. N. Dourdin, A.K. Bhatt, P. Dutt, P.A. Greer, J.S. Arthur, J.S. Elce, A. Huttenlocher, Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts, J. Biol. Chem. 276 (2001) 48382–48388. N.O. Carragher, B. Levkau, R. Ross, E.W. Raines, Degraded collagen fragments promote rapid disassembly of smooth muscle focal adhesions that correlates with cleavage of pp125 (FAK), paxillin, and talin, J. Cell Biol. 7 (1999) 619–630. N.O. Carragher, V.J. Fincham, D. Riley, M.C. Frame, Cleavage of focal adhesion kinase by different proteases during SRC- regulated transformation, J. Biol. Chem. 276 (2001) 4270–4275. A. Huttenlocher, S.P. Palecek, Q. Lu, W. Zhang, R.L. Mellgren, D.A. Lauffenburger, M.H. Ginsberg, A.F. Horwitz, Regulation of cell migration by the calcium-dependent protease calpain, J. Biol. Chem. 272 (1997) 32719–32722. A. Glading, P. Chang, D.A. Lauffenburger, A. Wells, Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway, J. Biol. Chem. 275 (2000) 2390–2398. A.M. Steff, S. Carillo, M. Pariat, M. Piechaczyk, Decreased susceptibility to calpains of v-FosFBR but not of v-FosFBJ or v-JunASV17 retroviral proteins compared with their cellular counterparts, Biochem. J. 323 (1997) 685–692. T. Hiwasa, N. Mitsuyuki, M. Nakata, S. Ohno, M. Maki, K. Suzuki, M. Takiguchi, Regulation of transformed state by calpastatin via PKCε in NIH3T3 mouse fibroblasts, Biochem. Biophys. Res. Commun. 290 (2002) 510–517. C. Braun, M. Engel, M. Seifert, B. Theisinger, G. Seitz, K.D. Zang, C. Welter, Expression of calpain I messenger RNA in human renal cell carcinoma: correlation with lymph node metastasis and histological type, Int. J. Cancer 84 (1999) 6–9. E. Shiba, J.I. Kambayashi, M. Sakon, T. Kawasaki, T. Kobayashi, H. Koyama, E. Yayoi, Y. Takatsuka, S.I. Takai, Ca2+ -dependent neutral protease (calpain) activity in breast cancer tissue and estrogen receptor status, Breast Cancer 3 (1996) 13–17. Y. Kimura, H. Saya, M. Nakao, Calpain-dependent proteolysis of NF2 protein: involvement in schwannomas and meningiomas, Neuropathology 20 (2000) 153–160.