The molecular makeup of spontaneous cancers at the clinical stage usually presents a complex mixture of primary and consequential events, some of which are thought to contribute to essential aspects of tumor progression (Hanahan and Weinberg, 2000
). Cancer syndrome associated mutations in contrast, often constitute well-defined primary tumorigenic events that are causal to tumor formation (Nagy et al., 2004
). Specifically, germ-line mutation of PTEN
has been shown to underlie the closely related autosomal dominant tumor susceptibility syndromes Cowden disease and Bannayan-Riley-Ruvalcaba syndrome (Eng, 2003
). Therefore, we anticipated that PTEN
germline mutations hold valuable information and novel insights into PTEN function in tumor initiation. While most of the described mutations result in truncation many PTEN missense mutations affect its catalytic activity. Through investigating instead how the enzymatically functional PTENK289E
mutant could be at the root of inheritable cancer, we have come to several novel conclusions:
I) We have discovered that deficiency in PTEN import causes cancer susceptibility, independent of catalytic activity. While it has been previously shown that the PTENK289E
mutant retains catalytic activity (Georgescu et al., 2000
), its nuclear import defect has so far remained elusive. Correlation of the K289E import defect with the observed cytoplasmic accumulation in the relevant Cowden patient tissue thus represents the first example of a causal relation between PTEN localization and cancer initiation. Since no such certainty on the cause-and-effect relation exists in sporadic mutations like PTENK13E
(which as published also suffers reduction in catalytic activity (Walker et al., 2004
)), the K289E mutation allows us to unambiguously ascribe a tumor suppressive function to PTEN nuclear localization. In agreement with this finding we find significant correlation between low tumor stage in colon cancer and dominant nuclear PTEN localization.
II) We show that nuclear PTEN import is regulated by mono-ubiquitination, which to our knowledge represents the first example of target protein ubiquitination enhancing nuclear import (and shuttling). PTEN poly-ubiquitination, in contrast leads to its cytoplasmic retention and degradation (Ciechanover, 2005
) and is therefore oncogenic in nature (see also(Wang et al., submitted). Thus PTEN mono-ubiquitination and subsequent nuclear import has a protective function, a highly relevant finding for the discussion of NEDD4-1 in tumorigenesis (see below). As mentioned, ubiquitin charging of some E2-ligases has been shown to regulate their nuclear import (Plafker et al., 2004
), and intriguingly de-ubiquitination of the Bcl-3 transcription factor has been recently shown to cause its cytoplasmic accumulation (Massoumi et al., 2006
). Yet, Bcl-3 cytoplasmic accumulation due to loss of nuclear (Ub-mediated) retention cannot be excluded, in contrast to PTEN where ubiquitination dictates the actual transport rates.
Mono-ubiquitination has previously been implied in p53 nuclear export (Li et al., 2003
; Lohrum et al., 2001
), but p53 also contains several well-characterized NLS and NES sequences that enhance transport (Michael and Oren, 2003
). PTEN in contrast, may rely entirely on the ubiquitination system for shuttling since no transferable NLS-like sequences have been demonstrated for it and we have shown that the K13 cluster does not rely on charge for import. It will be interesting to see if PTEN shares ubiquitination-specific import receptors with the above-mentioned cargoes.
III) We identify K13 and K289 as two major and conserved sites for PTEN ubiquitination and we show that their mutation leads to a constitutive shuttling defect that can be overcome by forced mono-ubiquitination. Importantly, PIP2
phospholipid binding and allosteric PTEN activation functions have also been ascribed to the basic patch containing K13 (for a review see (Leslie and Downes, 2004
)) and it will be interesting to see how these functions affect ubiquitination and vice versa
Since further sites on PTEN can also be ubiquitinated both in vitro and in vivo, we hypothesize that mono-ubiquitination efficiency is a limiting step of PTEN shuttling, and that ubiquitin proteases reverse this process and antagonize shuttling because the nuclear pool of PTEN is not quantitatively mono-ubiquitinated. Taken together, our data suggest a model (), in which cytoplasmic mono-ubiquitination of PTEN leads to either PTEN import or alternatively poly-ubiquitination and PTEN degradation (by NEDD4-1). Nuclear PTEN-Ub can shuttle back to the cytoplasm or after de-ubiquitination remains nuclear and protected from cytoplasmic degradation until it is again mono-ubiquitinated and exported.
IV) We demonstrate that PTEN nuclear localization is coupled to activity of the only known PTEN E3-ligase, NEDD4-1, which can both mono- and poly-ubiquitinated PTEN in vitro and in vivo. Since we here show that mono-ubiquitination is essential for PTEN function, Nedd4-1 effectively has both oncogenic (PTEN degradation) and tumor suppressive (PTEN shuttling) potential. Several scenarios how PTEN levels and localization are regulated can therefore be envisioned: (i) NEDD4-1 modification/ protein association could switch it from mono- to poly-ubiquitination mode, (ii) NEDD4-1 localization could dictate PTEN levels in a particular compartment (in poly-ubiquitination mode) and/ or transport out of the compartment (in mono-ubiquitination mode). In this respect it is interesting to note that although many cells show dominant cytoplasmic NEDD4-1, some cell lines and certain murine tissues show strong nuclear NEDD4-1 (Trotman and Pandolfi, unpublished). Significantly, the above implications for NEDD4-1 localization and activity apply equally to PTEN de-ubiquitinases, which we propose to be essential for control of PTEN residence times and stability within the nuclear and cytoplasmic compartments as depicted ().
V) We find that nuclear PTEN is not only protected but still able to antagonize AKT and cause apoptosis. These findings are in line with our observation of dominant nuclear PTEN localization in early stage but not advanced stage colon cancer. Together with our observations in the Cowden patient tissue, these data suggest that retention of the nuclear import capability of PTEN is a critical tumor-suppressive determinant and it will be interesting to see if other patient derived PTEN mutations also target PTEN import instead of catalytic activity.
Taken together, we have through an integrative approach, linked the genetics of a cancer syndrome with the biochemistry and cell biology of protein modification to demonstrate how ubiquitination regulates nuclear import and tumor progression through PTEN. We therefore propose that ubiquitin is a decisive regulator of this major tumor suppressor and that further defining this process is crucial to understanding and developing ubiquitination-related cancer therapies.