Calpain Protease Regulates Cellular Mechanics
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Calpain Protease Regulates Cellular Mechanics
Metastatic tumor progression, the process by which tumor cells disseminate from their primary site of development, is dependent upon the disruption of both cell-cell and cell-matrix adhesions. These in turn are governed by the multiprotein complexes of Cadherin-dependent adheren junctions and integrin-linked FA (Focal Adhesion) attachment sites. Disruption of these complexes leads to enhanced cell motility and invasion of the surrounding tissue matrix. Calpain -mediated proteolytic degradation of the Cadherin and FA complexes represent a mechanism for disassembly of these complexes (Ref.1).

Calpains are a highly conserved family of intracellular, non-lysosomal, calcium-dependent cysteine proteinases with numerous isoforms in organisms ranging from mammals to Drosophila melanogaster and Caenorhabditis elegans and with homologs in yeast and bacteria. They function in Ca2+ signaling by modulating biological activities of their substrates through limited proteolysis (Ref.2). Currently, at least 12 different Calpains have been identified in mammals with ubiquitous (such as m- and mu-Calpains) and tissue-specific (such as Calpain-3 (p94) and Calpain-4 in skeletal muscle) expression patterns. The m-Calpain, mu-Calpains and Calpastatin are the best-characterized members in the family (Ref.3). They are pivotal proteases participating in a limited proteolytic reaction of a number of cellular structural or regulatory proteins, such as cytoskeletal proteins, kinases, Cytokines, and the tumor-suppressing protein p53 (Ref.4). One of the best-documented functions for these enzymes is the regulation of integrin-mediated cell migration. Calpains release the link between the integrin-dependent FA complex and the actin cytoskeleton by proteolysis of talin, which allows proper cell migration (Ref.2). The activity of Calpains is tightly regulated by highly specific endogenous inhibitor Calpastatin (Ref.5). Inhibition of Calpain causes impaired retraction at the rear of the cell, an increase in the length of the tail at the retracting edge — which reflects suppressed de-adhesion and decreased cell movement. Furthermore, colocalization of Calpain-2 with FA structures including Src, paxillin, talin and Alpha-Actinin reside at cell adhesions and promotes their disassembly. This lead to reduced strength of integrin-mediated cell-matrix adhesions and enhanced cell migration (Ref.6).

Calpain-mediated proteolysis represents a major pathway of posttranslational modification of cellular proteins (Ref.7). Activation of v-Src induces the synthesis of Calpain, which promotes proteolytic cleavage of FAK (Focal Adhesion Kinase).FAK is then released from the cytoskeletal fraction into the cytoplasm, where it is further cleaved into fragments. So, Calpain-mediated cleavage of FAK results in dissociation of FAT (Focal-Adhesion-Targeting) sequence from the amino-terminal and kinase domains, and loss of FAK catalytic activity at FA (Ref.6). This disassembly abolishes the kinase activity of FAK at adhesion sites and impairs the ability of FAK to act as an adaptor molecule. This contributes to the disassembly of FA structures and results in the loss of Integrins, GF (Growth Factor) and Src -signaling to downstream effectors (Ref.6). During cell migration Calpain-1 (mu-Calpain) acts at the leading edge as a response to Integrin signals or calcium fluctuations due to the stress activated Ca2+ channels. Calpain-1 cleaves the target proteins, talin, ezrin, paxillin and the cytoplasmic tail of the Integrins to release the adhesion and form new adhesions. Calpain-2 (m-Calpain) is membrane bound and functions at the trailing edge of the migrating cell to cleave the Integrins in response to GFs receptor signals. PKA (Protein Kinase-A) functions to down regulate or inhibit Calpain-2. Calpain-1 in the Integrin clusters initially inactivates RhoA allowing the formation of lamellipodia. The subsequent activation of newly synthesized RhoA transforms these clusters into FA Complexes and the formation of contractile Actin -Myosin stress fibers. These mature adhesions do not contain Calpain . Signals derived from EGFR activation that are mediated through ERK(Extracellular Signal Regulated Kinase)/ MAPK (Mitogen-Activated Protein Kinase) enhance Calpain activity. Furthermore, inhibitors of Calpain suppress EGF-induced substrate de-adhesion and motility (Ref.6). Enhanced Calpain activity also accelerates the progression of transformed cells through the G1-phase of the cell cycle and is associated with hyperphosphorylation of Rb (Retinoblastoma protein) and increased protein levels of Cyclin-D, Cyclin-A and CDK2 (Cyclin-Dependent-Kinase). Calpains promote cleavage of CIK (CDK Inhibitor), P27(KIP1), which enhances the activity of G1 CDK, and results in hyperphosphorylation of Rb. So, Calpain provides a common link that regulates the ability of v-Src, and possibly c-Src, to promote both cell migration and proliferation (Ref.6). Increased levels of Calpain overcome the inhibitory action of Calpastatin and promote proteolytic degradation of Calpastatin, which further enhances Calpain activity.

Members of the Calpain family function in various biological processes including integrin-mediated cell migration, cytoskeletal remodeling, cell differentiation and apoptosis. At the pathological level, overactivation of Calpain as well as mutations abrogating Calpain activity have been implicated in cardiac and cerebral ischemia, platelet aggregation, neurodegenerative diseases, rheumatoid arthritis, cataract formation and LGMD2A (Limb Girdle Muscular Dystrophy-2A) (Ref.3). In human renal cell carcinomas, significantly higher levels of Calpain-1 expression are found in tumors that have metastasized to peripheral lymph nodes relative to tumors that apparently had not metastasized. Similarly, Calpain has been shown to play a role in the development of some schwannomas and meningiomas (Ref.1). Hyperactivation of Calpain due to elevated cellular Ca2+ levels contributes to tissue damage seen during ischemic injury to the heart and brain and during neurodegeneration. In Alzheimers disease, amyloid peptides induce aberrant Calpain activation that results in mislocalization of CDK5 due to proteolytic processing of the CDK5-related Cyclin p35 to Cyclin p25. Deregulated CDK5 hyperphosphorylates Tau, which promotes neuronal death. Administration of Calpain inhibitors lessens or prevents tissue damage, but the toxicity and lack of specificity of current inhibitors weakens the effectiveness of such therapies (Ref.2).