| pmid | 10866658 | ||||
|---|---|---|---|---|---|
| title | Phosphorylation of the PTEN tail regulates protein stability and function. | ||||
| authors |
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| journal | Mol Cell Biol | ||||
| year | 2000 | ||||
| full_text_available | true | ||||
| full_text_extraction_method | html | ||||
| pmcid | PMC85951 | ||||
| doi | 10.1128/MCB.20.14.5010-5018.2000 |
Authors: Vazquez F, Ramaswamy S, Nakamura N, Sellers WR Journal: Mol Cell Biol (2000) DOI: 10.1128/MCB.20.14.5010-5018.2000 PMC: PMC85951
- Mol Cell Biol. 2000 Jul;20(14):5010-8. doi: 10.1128/MCB.20.14.5010-5018.2000.
Phosphorylation of the PTEN tail regulates protein stability and function.
Vazquez F(1), Ramaswamy S, Nakamura N, Sellers WR.
Author information: (1)Department of Adult Oncology, Dana-Farber Cancer Institute, and Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
The PTEN gene is a tumor suppressor localized in the frequently altered chromosomal region 10q23. The tumor suppressor function of the PTEN protein (PTEN) has been linked to its ability to dephosphorylate the lipid second-messenger phosphatidylinositol 3,4, 5-trisphosphate and phosphatidylinositol 3,4-bisphosphate and, by doing so, to antagonize the phosphoinositide 3-kinase pathway. The PTEN protein consists of an amino-terminal phosphatase domain, a lipid binding C2 domain, and a 50-amino-acid C-terminal domain (the "tail") of unknown function. A number of studies have shown that the tail is dispensable for both phosphatase activity and blocking cell growth. Here, we show that the PTEN tail is necessary for maintaining protein stability and that it also acts to inhibit PTEN function. Thus, removing the tail results in a loss of stability but does not result in a loss of function because the resultant protein is more active. Furthermore, tail-dependent regulation of stability and activity is linked to the phosphorylation of three residues (S380, T382, and T383) within the tail. Therefore, the tail is likely to mediate the regulation of PTEN function through phosphorylation.
DOI: 10.1128/MCB.20.14.5010-5018.2000 PMCID: PMC85951 PMID: 10866658 [Indexed for MEDLINE]
Abstract
The PTEN gene is a tumor suppressor localized in the frequently altered chromosomal region 10q23. The tumor suppressor function of the PTEN protein (PTEN) has been linked to its ability to dephosphorylate the lipid second-messenger phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate and, by doing so, to antagonize the phosphoinositide 3-kinase pathway. The PTEN protein consists of an amino-terminal phosphatase domain, a lipid binding C2 domain, and a 50-amino-acid C-terminal domain (the “tail”) of unknown function. A number of studies have shown that the tail is dispensable for both phosphatase activity and blocking cell growth. Here, we show that the PTEN tail is necessary for maintaining protein stability and that it also acts to inhibit PTEN function. Thus, removing the tail results in a loss of stability but does not result in a loss of function because the resultant protein is more active. Furthermore, tail-dependent regulation of stability and activity is linked to the phosphorylation of three residues (S380, T382, and T383) within the tail. Therefore, the tail is likely to mediate the regulation of PTEN function through phosphorylation.
DISCUSSION
Recently it was shown that PTEN;1-351 has in vitro lipid phosphatase activity, can inhibit Akt activation, and can suppress anchorage-independent growth ( 14 ). In accordance with these results, we found that PTEN;1-353 inhibits the growth of PTEN null 786-0 cells in soft agar and induces apoptosis in LNCaP cells (S. Ramaswamy and W. R. Sellers, unpublished data). Furthermore, the PTEN crystal structure shows that these residues include the entire PD and a C2 lipid binding domain ( 22 ). Together these data suggest that the tail (residues 354 to 403) is not required for biological activity of the protein.
Here, we have shown that deletion of the PTEN tail results in a loss of protein stability. One might expect that loss of stability would lead to a loss of PTEN function; however, as stated above, no requirement for the tail was found in multiple assays of PTEN function. This paradox can be explained by the concomitant loss of an inhibitory activity that maps to the tail region. Thus, the tail contains sequences that are required for protein stability and for negative regulation of PTEN function. This linkage between stability and activity argues for a model in which PTEN is normally found in a stable yet relatively inactive state. In such a model, activation of PTEN would be accompanied by a decrease in protein half-life, presumably reflecting degradation of the active molecule (Fig. 7 ). Such a mechanism would prevent the untimely or promiscuous activation of PTEN. The linkage between protein activation and protein instability is a common theme in molecular biology. Examples of proteins where the activated form of the protein is unstable include Src, EGFR, PDGFR, and the nuclear receptors RAR and RXR ( 16 , 31 , 47 ).
The tail is also required for PTEN phosphorylation. In vitro mutagenesis revealed specific phosphorylation of S380, T382, and T383. Mutation of these sites led to a loss of stability and a gain in PTEN function. Conversely, aspartic acid mutations of these same residues preserved the protein half-life and the function of PTEN. Together, these data argue strongly that phosphorylation of these residues is required for stability and that the changes in stability are not simply the result of a misfolding secondary to changes in the amino acid residues. Recently others have shown that mutations in the C2 domain can reduce expression levels and protein half-life ( 14 ). The mutants are, however, found to be functionally inactive or significantly impaired in biological assays; thus the instability of these mutants might indeed arise as a consequence of protein misfolding ( 14 , 22 ). On the other hand, the PTEN;A3 mutant, while unstable, is more active, arguing strongly against protein misfolding as a mechanism for instability in this instance.
What is the mechanism by which PTEN phosphorylation regulates protein stability? As pointed out by Georgescu et al. ( 14 ) the tail contains two putative PEST sequences (residues 350 to 375 and 379 to 386) implicated in targeting proteins for proteolytic degradation. We have found that the second PEST region is the site of three phosphorylation sites (S380, T382, T383), raising the possibility that this sequence is normally masked by phosphorylation. Arguing against this model, however, is the fact that deletion of the entire tail also results in a shorter half-life. Secondary structure prediction and the results of proteolytic digestion experiments suggest that the tail is a relatively unstructured and presumably flexible region. Thus, one model is that the tail can mask a degradation signal present elsewhere in the PTEN protein when phosphorylated; this signal would be unmasked by dephosphorylation leading to a shift in the position of the tail (Fig. 7 ).
As an alternative model, the tail might regulate PTEN localization through interactions with the adjacent C2 domain. If so, then stability might simply reflect the localization of PTEN to a subcellular compartment where PTEN degradation can take place. To date we have not seen an obvious effect on the membrane localization of the relevant C-terminal truncations or phosphorylation site mutants (data not shown). Surprisingly, PTEN;1-353 and PTEN;A4 manifestly increased localization to the nucleus (data not shown). Whether this apparent change in localization results from a more rapid degradation of the cytoplasmic component of these mutants or from a true shift in localization is not yet clear. Nonetheless, regulation of localization by tail phosphorylation might play an important role in the regulation of PTEN.
What is the mechanism through which PTEN function is regulated by the tail? There are a number of mechanisms that could account for the inhibitory activity of the tail on PTEN. First, the phosphatase activity itself could be regulated through an allosteric or steric mechanism. To date, however, we have seen no effects of phosphorylation site mutations on intrinsic phosphatase activity (data not shown). Second, the ability of PTEN to gain access to a substrate could be altered either through changes in localization (see above), through changes in the position of the tail, or perhaps through inhibitory interactions with tail-associated proteins. With respect to the last idea, the PTEN tail contains a PDZ binding domain. Given that the PDZ binding sequence (along with the entire tail) is dispensable for the biological function of PTEN, it would seem likely that this domain is linked to the role for the tail that we have put forth. Specifically, the interaction of a PTEN with a PDZ domain-containing protein might be regulated through PTEN phosphorylation.
Are the PTEN phosphorylation events that we have identified constitutive or regulated? Our data are most consistent with the idea that PTEN exists in a predominantly phosphorylated state and that dephosphorylation of the S380 cluster is a regulated event. While PTEN runs as a single band under standard sodium dodecyl sulfate-gel electrophoresis conditions, on two-dimensional gels multiple isoforms of PTEN can be distinguished based on differences in the isoelectric point (data not shown). While it is likely that these forms represent different PTEN phosphoisoforms, how these forms are related to each phosphorylation site or to PTEN activity or stability is not yet known. Our model suggests that PTEN function is regulated by the balance between a kinase and a phosphatase. It is possible that the kinase constitutively phosphorylates PTEN and that a phosphatase regulates the activity. An intriguing possibility is that PTEN activates itself through autodephosphorylation maintaining a constant loop of activity. Identification of the kinase and phosphatase activities responsible for the regulation of these sites should provide further insights into how regulation of PTEN is achieved.