The PTEN tail modulates PTEN stability.
Recently the crystal structure of PTEN has been solved from residues 7 to 353 (eliminating an internal loop of residues 286 to 309). This truncated protein has in vitro lipid phosphatase activity comparable to that of full-length PTEN (PTEN;WT) and can induce apoptosis in LNCaP cells to the same extent as the wild type (14
). Similarly, our group mapped the minimal in vivo functional domain of PTEN by C-terminal and N-terminal deletion mutations (S. Ramaswamy and W. R. Sellers, unpublished data). We found that a truncated PTEN protein of residues 10 to 353 retained protein and lipid phosphatase activity in vitro and was able to induce a G1
arrest in cells. Furthermore, PTEN;1-353 was comparable to PTEN;WT in suppressing soft-agar colony formation in PTEN null renal carcinoma cells (786-0 cells) (S. Ramaswamy and W. R. Sellers, unpublished data). These results indicate that the last 50 amino acids of PTEN are not necessary for lipid or protein phosphatase activity or for its ability to inhibit proliferation or induce apoptosis. For simplicity we refer to these last 50 residues as the PTEN tail (Fig. A).
FIG. 1 The PTEN tail is required for protein stability. (A) Schematic representation of the PTEN protein. Dark gray box, phosphatase signature motif (HCXXGXXR); light gray box, PD; white box, C2 domain; black box, C-terminal 50-residues (PTEN tail). The minimal (more ...)
In our experiments we noted that PTEN;1-353 was produced at markedly reduced steady-state levels compared to PTEN;WT. To determine whether the changes in the steady-state protein levels were related to changes in protein stability, 786-0 cells (PTEN null) transiently transfected with plasmids encoding PTEN;WT or PTEN;1-353 were pulse-labeled with [35
S]methionine for 45 min. HA-PTEN and HA-PTEN;1-353 were recovered by immunoprecipitation and detected by autoradiography. In these experiments the half-life of PTEN;1-353 was found to be reduced by more than fourfold compared to that of PTEN;WT (Fig. B and C). In keeping with these results, it was recently reported that the steady-state level of PTEN;1-351 is reduced compared to that of PTEN;WT when the protein is produced by transfection in COS-7 cells (14
). Together these data suggest that the PTEN tail, while not required for the functional activity of the protein, is required for maintaining stability.
The tail domain modulates PTEN biological activity.
As a result of the reduced half-life, PTEN;1-353 is produced at significantly lower levels than PTEN;WT. In order to determine whether the resulting decrease in protein production results in reduced activity, PTEN;1-353 and PTEN;WT were compared in a cell cycle arrest assay that reflects the ability of PTEN to act as a lipid phosphatase (39
). 786-0 cells were transfected with increasing doses of plasmids encoding PTEN;WT and PTEN;1-353 along with a plasmid encoding the cell surface marker pCD19. The cell cycle distribution of the CD19-positive cells (as a marker of transfection) was determined by staining with fluorescein isothiocyanate-conjugated anti-CD19 and propidium iodide followed by two-color FACS. Surprisingly, at equivalent input plasmid concentrations PTEN;1-353 reproducibly induced a greater increase in G1
than PTEN;WT (data not shown). Next, the activities of PTEN;WT and PTEN;1-353 were compared when the proteins were produced at similar steady-state levels. Plasmid titration indicated that equivalent protein levels were obtained at 2 μg of PTEN;1-353 and 0.5 μg of PTEN;WT (Fig. A and B). At these levels PTEN;1-353 induced a significantly more robust G1
arrest (Fig. C).
FIG. 2 The PTEN tail is an inhibitory domain. (A) Steady-state protein levels of PTEN;WT and PTEN;1-353. 786-0 cells were transfected with the indicated amounts of plasmids encoding HA-PTEN;WT or HA-PTEN;1-353. Forty hours after transfection, cell lysates were (more ...)
Forkhead transcription factors are targets of Akt regulation both in mammalian cells and in Caenorhabditis elegans
). Akt phosphorylation creates 14-3-3 binding sites and results in the cytoplasmic localization of forkhead proteins. Furthermore, recent data in our laboratory have shown that in PTEN null cells FKHRL1 and exogenously expressed FKHR are retained in the cytoplasm and that exogenously expressed FKHR fails to activate transcription. Coexpression of wild-type PTEN, but not a PTEN mutant lacking lipid phosphatase activity, with FKHR restores cytoplasmic localization and FKHR transactivation as measured with a 3XIRS promoter or FasL promoter luciferase reporters (Nakamura et al., submitted). Thus, FKHR transcriptional activity requires PTEN function. The induction of FKHR transcriptional activity by PTEN is also dose dependent, as shown in Fig. D.
We next compared PTEN;WT and PTEN;1-353 in a FKHR transcriptional activation assay. Consistent with the results obtained in the cell cycle assay (Fig. C), the ability of PTEN;1-353 to induce FKHR transcriptional activity was enhanced compared to that of PTEN;WT. Furthermore, at every DNA plasmid concentration tested, PTEN;1-353 induced FKHR activation more efficiently than PTEN;WT, although protein levels were reduced by more than fourfold. These results suggest that the PTEN tail not only plays a role in maintaining its protein stability but also in regulating its biological activity. Specifically, these data suggest that the tail acts to restrict or inhibit PTEN function.
PTEN is a phosphoprotein.
The PTEN tail is rich in serine and threonine (28% of the residues) and contains consensus phosphorylation sites for GSK3, PKA, CK1, and CK2. In order to determine whether regulation of PTEN stability and activity might be linked to phosphorylation, we determined whether endogenous PTEN is phosphorylated. To this end, ACHN renal carcinoma cells that contained PTEN were metabolically labeled with [32P]orthophosphate. Labeled lysates were incubated with an anti-PTEN antibody (C54) or a preimmune control. Bound proteins were separated by gel electrophoresis and detected by autoradiography. A 32P-labeled protein of the same molecular weight as PTEN was detected in anti-PTEN immunoprecipitates but not in the preimmune control (Fig. A). In separate experiments in which the labeled proteins were transferred to nitrocellulose, this 32P-labeled species comigrated with PTEN, as detected by immunoblotting. These data suggest that endogenous PTEN is phosphorylated. Next, PTEN plus U2-OS cells were transfected with either the empty vector (pSG5L) or pSG5L-HA-PTEN and metabolically labeled with orthophosphate. Labeled cells were lysed, and epitope-tagged proteins were immunoprecipitated with anti-HA antibody. In parallel, endogenous PTEN was immunoprecipitated from lysates prepared from untransfected orthophosphate-labeled U2-OS cells. A phosphorylated protein of 58 kDa was detected in the anti-HA immunoprecipitates from cells expressing HA-PTEN but not in the vector-transfected cells (Fig. B). Next, phosphorylated endogenous PTEN and exogenously produced HA-PTEN were digested with trypsin. As shown in Fig. C, an identical pattern of phosphotryptic peptides was observed when phosphopeptides were separated by Tris-Tricine gel electrophoresis (16.5% acrylamide). Phosphoamino acid analysis of both endogenous and transfected PTEN proteins showed phosphorylation of serine and threonine residues, while tyrosine phosphorylation was not detected (Fig. D). These data suggest that endogenous PTEN is a phosphoprotein, that HA-PTEN produced by transfection is a phosphoprotein, and that these proteins are phosphorylated on the same peptides, predominantly on serine and threonine.
FIG. 3 PTEN is a phosphoprotein. (A) Phosphorylation of endogenous PTEN. Asynchronously growing ACHN cells were metabolically labeled with [32P]orthophosphate for 4 h. Protein extracts were prepared and immunoprecipitated with either preimmune (more ...) PTEN is phosphorylated within the tail domain.
To determine the exact sites of phosphorylation, U2-OS cells were transfected with plasmids encoding a series of PTEN C-terminal deletion mutants and labeled with orthophosphate. 32P-labeled HA-tagged proteins were recovered by anti-HA immunoprecipitation and detected by autoradiography. These experiments revealed that deletion of residues 354 to 403 (the tail) abrogated most PTEN phosphorylation (Fig. A). In addition, gel electrophoretic separation of peptides generated by cyanogen bromide cleavage of orthophosphate-labeled HA-PTEN revealed a single phosphorylated peptide consistent in size with the predicted CNBr peptide containing the PTEN tail (data not shown). Next, every serine and threonine in the tail was mutated either singly or in clusters to alanine. These PTEN mutants were transfected into U2-OS cells and labeled with orthophosphate. No single-amino-acid substitution abrogated or significantly reduced the total phosphorylation of PTEN (data not shown). However, the substitution of a serine/threonine cluster, S380, T382, T383, and S385 (the A4 mutant), did significantly alter total PTEN phosphorylation. In addition, mutation of these sites led to the loss of the more slowly migrating tryptic phosphopeptide (Fig. B). This peptide therefore is likely to be peptide 2 (Fig. D). In contrast, substitution of alanines for the serine/threonine cluster beginning at S360 (which contains a GSK3 consensus phosphorylation site) had no effect on either the total phosphate incorporated into PTEN or the phosphorylation of the two phosphopeptides detected in Tris-Tricine gels (Fig. B). Next, the A4 mutation was combined with a single alanine substitution at a consensus CK2 site (S370) found in peptide 1 of the tail to give the A5 mutation (Fig. D). When PTEN;A5 was expressed in U2-OS cells and tested for phosphorylation, 32P labeled protein was not detected despite adequate protein expression (Fig. C). Phosphopeptide analysis was not possible because no labeled protein could be excised from the gel. These results indicate that most, if not all, of the PTEN tail phosphorylation occurs on serine 370 and one or more sites of the A4 cluster (S380, T382, T383, and S385).
FIG. 4 PTEN is phosphorylated within the tail. (A) Deletion of the tail impairs PTEN phosphorylation. Plasmids encoding either wild-type (WT) HA-PTEN or the indicated C-terminal truncation mutants were transfected into U2-OS cells. Twenty-four hours after transfection, (more ...)
To directly identify the PTEN phosphorylation sites, 2 μg of HA-PTEN isolated by anti-HA immunoaffinity purification was digested with trypsin and analyzed by LC/MSMS. Here, peptide 1 with a phosphoserine at residue 370 was identified. However, peptide 2 was not detectable in either a phosphorylated or unphosphorylated state. No other phosphopeptides were identified (data not shown).
Mutation of the phosphorylation sites in the tail alter PTEN stability and biological activity.
To determine whether phosphorylation of one or more of the amino acid residues delineated above has a role in modulating PTEN stability, the relevant phosphorylation site mutants were produced in U2-OS cells by transient transfection and the steady-state levels of HA-PTEN and the phosphorylation mutants were determined by immunoblot analysis. While mutation of serine 370 did not change the steady-state level of PTEN (data not shown), mutation of the S/T cluster (PTEN;A4) resulted in a marked decrease in the steady-state protein level (data not shown). Furthermore, pulse-chase labeling experiments revealed a marked reduction in the PTEN;A4 half-life (Fig. A, left).
FIG. 5 Mutation of phosphoacceptor sites in the PTEN tail alters protein stability and activity. (A) Protein half-life of PTEN;A4 (left) and single-substitution mutants (middle and right). 786-0 cells were transfected with plasmids encoding HA-PTEN;WT and HA-PTEN;A4 (more ...)
As deletion of the tail led to an increase in PTEN activity, we next asked if the PTEN;A4 mutant was similarly more active in biological assays. In keeping with the data for the PTEN tail, the PTEN;A4 mutant, while expressed at lower levels, was more active in both inducing a G1 arrest in PTEN null 786-0 cells (Fig. B, left) and inducing FKHR transcriptional activation (Fig. C, left). These data suggest that phosphorylation within the A4 cluster is required to maintain stability and is linked to an inhibitory activity of the PTEN tail.
Next, the individual point mutations within the A4 cluster were tested in the same assays of protein half-life and activity. While replacement of serine 385 with alanine did not alter the steady-state PTEN protein levels, mutation of S380, T382, and T383 each reduced both the steady-state protein levels and the protein half-life (Fig. A, right, and data not shown). More specifically, the half-life of the PTEN;A4 mutant was reduced more than six-fold compared to that of PTEN;WT. Similarly, mutating serine 380 reduced the half-life by more than fivefold. Mutation of threonines 382 and 383 reduced PTEN half-life by 2.7- to 3-fold. Furthermore, the individual phosphorylation mutants with substitutions S380A, T382A, and T383A, but not S385A, were again more active in inducing a G1 arrest and in inducing FKHR transcriptional activation (Fig. B and C, right). Taken together, these data show that the increased activity associated with deletion of the tail is entirely mimicked by mutations within the A4 cluster, specifically S380, T382, or T383.
The above data raised the possibility that phosphorylation of these three specific residues (S380, T382, and T383) might be required to maintain PTEN in a stable yet relatively inactive state. While mutation of each of these individual residues did not alter total incorporation of 32P into the PTEN protein (data not shown) when the S380, T382, and T383 residues were mutated to alanine (PTEN;A3) incorporation of 32P into PTEN during orthophosphate labeling was reduced (Fig. A) and the most slowly migrating tryptic phosphopeptide (peptide 2) was not detectable (Fig. B). In keeping with the data for PTEN;A4 and for the individual phosphorylation site mutants (with mutations S380A, T382A, and T383A), PTEN;A3 was found to have a reduced protein half-life, to be expressed at lower steady-state levels, and to be more active than wild-type PTEN in biological assays (Fig. C to F).
FIG. 6 Aspartic acid substitutions of serine 380, threonine 382, and threonine 383 restore PTEN expression levels and half-life. (A) Substitution mutations of serine 380, threonine 382, and threonine 383 impair PTEN phosphorylation. Plasmids encoding PTEN;WT (more ...) Aspartic acid substitutions at the phosphorylation sites in the PTEN tail lead to a recovery of PTEN stability.
A reasonable interpretation of these results is that the serine/threonine-to-alanine substitutions of PTEN block phosphorylation and thereby alter the stability and activity of PTEN in cells. On the other hand, it is formally possible that mutation of these residues might result in these changes independent of the changes in PTEN tail phosphorylation. To distinguish these possibilities, conversion of the serines/threonines to aspartic acid was used to try and mimic phosphorylation of these residues. As we had noted that mutation of any one of the putative phosphorylation sites altered both stability and activity, it appeared that phosphorylation of all three residues might be required for maintaining PTEN stability. Therefore, we generated a PTEN;D3 cDNA in which codons 380, 382, and 383 encoded aspartic acid.
In contrast to the results obtained with PTEN;A3, PTEN;D3 recovered the expression levels of PTEN;WT, suggesting that a negative charge was enough to maintain protein stability (Fig. C). To test this possibility, we performed a pulse-chase experiment to determine if aspartic acid substitution could restore PTEN stability. As shown in Fig. D, PTEN;D3 recovered the stability of PTEN;A3 and was similar to PTEN;WT. As expected, when analyzed after orthophosphate labeling, PTEN;D3 like PTEN;A3 was found not to contain phosphopeptide 1 (data not shown). These data argue that the D3 mutation does not restore stability simply be restoring phosphorylation of PTEN at other sites but rather that phosphorylation of the S380 cluster is required for appropriate stability.
Next, we asked whether aspartic acid substitution led to a change in the activity of PTEN in the cell cycle assay and the FKHR transactivation assay. As would be predicted if loss of phosphorylation was responsible for the changes seen in PTEN activity, PTEN;D3 recovered the activity of PTEN;WT and its activity was reduced compared to that of PTEN;A3 (Fig. E-F).