Phosphorylation-regulated Cleavage of the Tumor Suppressor PTEN by Caspase-3

Abstract

PTEN phosphatase is one of the most commonly targeted tumor suppressors in human cancers and a key regulator of cell growth and apoptosis. We have found that PTEN is cleaved by caspase-3 at several target sites, located in unstructured regions within the C terminus of the molecule. Cleavage of PTEN was increased upon TNF␣-cell treatment and was negatively regulated by phosphorylation of the C-terminal tail of PTEN by the protein kinase CK2. The proteolytic PTEN fragments displayed reduced protein stability, and their capability to interact with the PTEN interacting scaffolding protein S-SCAM/MAGI-2 was lost. Interestingly, S-SCAM/ MAGI-2 was also cleaved by caspase-3. Our findings suggest the existence of a regulatory mechanism of protein stability and PTEN-protein interactions during apoptosis, executed by caspase-3 in a PTEN phosphorylationregulated manner.The PTEN gene, one of the tumor suppressor genes most frequently mutated in human cancers, encodes a protein of 403 amino acids with phosphatase activity (1-4). PTEN contains tyrosine phosphatase activity toward phosphorylated peptides and proteins in vitro (5, 6). It has been proposed that PTEN controls cell motility and invasiveness by tyrosine dephosphorylation of the focal adhesion kinase, Fak, and the adapter protein Shc (7). On the other hand, PTEN possesses lipid phosphatase activity toward 3-phosphoinositides, counteracting the action of the oncogenic phosphatidylinositol 3-kinases and blocking the activation of the proto-oncogene protein kinase B/Akt (8, 9). Structurally, PTEN is composed of two domains, an N-terminal phosphatase catalytic domain and the C-terminal C2 domain, which binds phospholipids in vitro and is thought to mediate the binding of the molecule to membranes (10). In addition, PTEN contains three unstructured regions: first, the first seven residues form part of a phosphatidylinositol (4,5)-diphosphate binding motif; second, an internal loop within the C2 domain (residues 286 -310) of unknown function; third, a C-terminal tail (residues 354 -403) that contains a region phosphorylated by the protein kinase CK2, as well as a PDZ binding motif located at the far C terminus (1-4). We and others have reported that the phosphorylation of PTEN by CK2 regulates its function and protein stability to proteasome-mediated degradation (11-13). The C-terminal PDZ binding motif of PTEN has been shown to interact with the second PDZ domain of the scaffolding proteins S-SCAM/MAGI-2 and MAGI-3. This interaction has been shown to enhance the inhibitory effect of PTEN on protein kinase B activation (14,15).Caspases (cysteinyl-directed aspartate-specific proteases) are a family of highly specific proteases that play a key role during the apoptotic cell death. Caspases, constitutively expressed as inactive zymogens, are activated in a proteolytic cascade, in which initiator caspases, such as caspase-8, are activated by autoproteolysis in response to apoptotic cell stimulation. Initiator caspases then proteolytically activate downstream executioner caspa...

Binding of PTEN to Specific PDZ Domains Contributes to PTEN Protein Stability and Phosphorylation by Microtubule-associated Serine/Threonine Kinases

“…Plasmids and Antibodies-pRK5 HA-PTEN (N-terminal tagging) and pGEX-4T PDZ-2/MAGI-2, pGEX-4T PTEN, and pCMV Myc-MAGI-2 (pCMV Myc-S-SCAM) have been described previously (16,33). The plasmids pGEX-4T PDZ-2/MAGI-3 (residues 567-697), pGEX-4T PDZ-2/hDlg (residues 308 -411), pGEX-4T PDZ-2/pDlg (residues 285-384), pGEX-4T PDZ/MAST205 (residues 1031-1138), pGEX-4T PDZ/ MAST3 (residues 939 -1046, DPS .…”

“…Binding of PTEN to PDZ Domains Increases PTEN Protein Stability-The C-terminal region of PTEN is important to maintain PTEN protein stability (13)(14)(15)(16)39), suggesting a possible stabilization of PTEN by binding to PDZ domain-containing proteins. To test this hypothesis, pulse-chase experiments were performed using the single amino acid substitution mutant K402W, whose association with PDZ domains is diminished (see Fig.…”

“…In addition, some PTEN biological functions have been attributed to its protein phosphatase activity (7)(8)(9)(10), and a PTEN phosphatase independent effect on the regulation of p53 stability and transcriptional activity has been reported (11). A major level of regulation of PTEN functions is related with its phosphorylation status, which has been involved in maintaining PTEN protein stability and in the control of PTEN subcellular location and/or its association with regulatory molecules (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). In this regard, PTEN possesses a C-terminal tail (last 54 amino acids; residues 350 -403), which harbors at its far C terminus a functional PDZ domain-binding motif (residues Thr 401 -Lys 402 -Val 403 -COOH).…”

Nuclear PTEN: a tale of many tails

“…In contrast, the nuclear exclusion region of the C-terminal tail (residues 368-390) (Gil et al 13 and our unpublished observations) is enriched in negative-charge residues, including Asp residues (some of which are targeted by caspase-3) and Ser and Thr-phosphorylated residues (Figure 1e). 20 Figure 2 shows the location of the nuclear exclusion motifs at the PTEN core surface (RKK-PTP, CBR3 and Ca2) and of the PTEN N-terminus (Arg 14 residue) in its tertiary structure. It has to be mentioned that the PTEN crystal lacks a portion of the PTEN N-terminal tail (residues 1-13), as well as the entire PTEN C-terminal tail (residues 350-403), making impossible the local assignment of these regions.…”

PCAF Modulates PTEN Activity

“…Structurally, the PTEN protein consists of an amino-terminal phosphatase domain and a carboxyl-terminal domain, which is subdivided into C2, phosphorylation, and PDZ binding domains. The C2 domain lends itself to regulation of PTEN function by contributing to membrane localization, and the phosphorylation domain controls protein stability as governed by casein kinase II-directed phosphorylation (11)(12)(13). Given the constitutive nature of casein kinase II activity, other more tightly controlled mechanisms of regulation of PTEN function would be predicted.…”