April Newsletter: Posttranslational Regulation of Phosphatase and Tensin Homolog (PTEN)

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Introduction

Worldwide, more than 47 million people have been diagnosed with dementia, and the majority of these cases are caused by Alzheimer’s disease (AD); aside from the social burden, this neurodegenerative disease has an associated cost of 1.09% of the global gross domestic product1. Severe cognitive impairment that leads to deficits in skilled movements, language, and recognition are pathophysiological hallmarks of AD. On a molecular level, neuropathological hallmarks include formation of beta-amyloid plaques and neurofibrillary tangles (NFTs) comprised of paired helical filaments of hyper-phosphorylated Tau proteins. This newsletter focuses on the mechanistic control of Tau by post-translational modifications (PTMs) and the development of novel AD therapeutics based on regulating the PTM status of Tau2 (Fig. 1).

Tau: Phosphorylation

Increased levels of total Tau in the cerebral spinal fluid (CSF) are associated with AD and other neurodegenerative diseases; thus, total Tau levels have been defined as a marker for neuronal damage and degeneration rather than a specific marker for AD3. Conversely, increased levels of tau phosphorylated at T181 in CSF are uniquely linked to AD, making it one of the few specific biomarkers for AD diagnosis. For confirmation of AD pathology post-mortem, NFTs are routinely used, and recent evidence suggests that tau oligomers are detectable at early Braak stages of AD; furthermore, NFT maturation and distribution correlate with cognitive decline in AD, suggesting that NFTs have a critical role in AD pathogenesis4.

Figure 1. Domains of PTEN and PTMs within domains at specific sites. PBD, PIP2 binding domain. Adapted from reference #19.
Figure 1: PTMs of Tau being targetedas Alzheimer's disease therapeutics.

 

The PTM phosphorylation regulates physiological Tau interactions with microtubules4, but in AD, Tau is hyper-phosphorylated, which is a driving, regulatory mechanism in Tau mislocalization, dysfunction, aggregation, and NFT formation5,6. Tau hyper-phosphorylation can occur on as many as 45 residues, some of which are distinct from normal Tau phosphorylation sites3,6. Tau hyper-phosphorylation is regulated by several kinases including cyclic-AMP-dependent protein kinase, c-Jun N-terminal kinase 3 (JNK3), glycogen synthase kinase 3 beta (GSK3B), and cyclin dependent kinase 5 (CDK5), among others2. JNK3, CDK5, and GSK3B inhibitors have all demonstrated neuroprotective properties in vitro and in animal models. GSK3B inhibitors have been tested in clinical trials, but no significant benefit was observed, leading to termination of these studies2,7. However, inhibition of JNK3, which was upregulated in CSF along with CDK5, may still be a viable therapy, and further investigation is ongoing. An alternative approach to regulating Tau hyper-phosphorylation is to activate the phosphatase that dephosphorylates Tau. Sodium selenite, an agonist for protein phosphatase 2 (PP2A), is in development and produces cognitive improvements in AD mouse models8,9.  

Tau: Ubiquitination and SUMOylation

The PTMs ubiquitination and SUMOylation have also been identified as key regulators of Tau activity and NFT formation. The ubiquitin ligase, C-terminus of Hsc70-interacting protein (CHIP), offered significant protection against NFT formation in a mouse tauopathy model. This finding complements the inverse relationship between CHIP and pathogenic Tau in AD brains and provides evidence that ubiquitination may be an essential clearance mechanism for aggregated Tau10. A recent study by Luo et al. identified significant crosstalk between Tau hyper-phosphorylation and Tau SUMOylation, where either modification enhanced the other11. Moreover, SUMOylation of Tau prevented poly-ubiquitination and subsequent Tau degradation, possibly leading to aggregation. This study highlighted the significant crosstalk between different PTMs of Tau, and may provide a rationale to target alternative PTM regulatory mechanisms of Tau to ultimately regulate the hyper-phosphorylation of Tau. Indeed, PTMs that crosstalk with hyper-phosphorylation are not confined to ubiquitination and SUMOylation. Crosstalk between Tau hyper-phosphorylation and glycosylation is a well-characterized mechanism of regulation12,13.

Tau: Acetylation

Recently, acetylation was identified as a Tau PTM elevated at early Braak stages of AD and shown to positively regulate hyper-phosphorylated Tau levels and Tau aggregation in vitro14,15. Deleting the deacetylase SIRT 1 elevated Tau acetylation, which suppressed poly-ubiquitination and subsequent protein turnover, providing additional evidence that Tau acetylation promotes AD progression14. These findings further highlight the importance of PTM crosstalk in Tau regulation. Building upon these studies, Min et al. identified K174 as the specific lysine critical for Tau acetylation, and defined the lysine acetyltransferase P300 as a regulator of Tau acetylation16. K174 was acetylated in early and late Braak stages of AD. Importantly, the prescription drug, salsalate, which decreases P300 activity, reversed Tau-mediated memory impairments and hippocampal atrophy in a mouse tauopathy model. Importantly, when salsalate was used on neurons expressing a Tau lysine acetylation mimetic, K174Q, it provided no significant benefit as shown by unchanged levels of total and phosphorylated Tau and atrophy of the hippocampus. These mutagenesis data provided additional evidence for a key role of acetylated Tau in AD progression. As this drug is already FDA approved, it will be interesting to see if it has the same benefits in treating patients with AD.

Conclusions

Pursuit of emerging AD therapeutics and subsequent drug development based on the mechanistic understanding of how PTMs regulate Tau is not an isolated event, as many pathological proteins in cancer, cardiovascular, metabolic, and other neurological diseases have dysfunctional post-translational regulation17-19. For example, tyrosine kinase receptors are often deregulated in cancer, and several viable cancer drugs work by controlling the receptors’ ability to induce downstream PTM signaling20. Identification of novel regulatory PTMs for pathological proteins may aid in the development of effective, targeted therapeutics. In addition, PTM crosstalk is a fundamental mechanism to control a target protein’s function.  Having the right tools to identify one or more novel PTMs for a target protein will be essential to gain a complete picture of how a target protein is regulated. To assist scientists in PTM studies, Signal Seeker™ kits offer unprecedented ability to measure endogenous levels of various PTMs of target proteins in a sensitive and quantitative manner.


Related Products & Resources

Signal Seeker™ Kits

Signal-Seeker™ Phosphotyrosine Enrichment Kit (Cat. # BK160)

Signal-Seeker™ SUMO 2/3 Enrichment Kit (Cat. # BK162)

Signal-Seeker™ Ubiquitin Enrichment Kit (Cat. # BK161)

Actin Biochem Kits

Phosphotyrosine Affinity Beads (Cat. # APY03-beads)

SUMO 2/3 Affinity Beads (Cat. # ASM24-beads)

Ubiquitin Affinity Beads (Cat. # UBA01-beads)

Control for Ippt IgG Beads (Cat. # CIG01-beads)

Control for Ubiquitin Affinity Beads (Cat. # CUB02)


References

  1. Shi Y. et al. 2012. PTEN at a glance. J. Cell Sci. 125, 4687–4692.
  2. Lee J.O. et al. 1999. Crystal structure of the PTEN tumor suppressor: Implications for its phosphoinositide phosphatase activity and membrane association. Cell. 99, 323-324.
  3. Song M.S. et al. 2012. The functions and regulation of the PTEN tumor suppressor. Nat. Rev. Mol. Cell Biol. 13, 283-296.  
  4. Al-Khouri A.M. et al. 2005. Cooperative phosphorylation of the tumor suppressor phosphatase tensin homologye (PTEN) by casein kinases and glycogen synthase kinase 3beta. J. Biol. Chem. 280, 35195-35202.
  5. Torres J. and Pulido R. 2001. The tumor suppresspr PTEN is phosphorylated by the protein kinase CK2 at its C terminus. Implications for PTEN stability to proteasome-mediated degradation. J. Biol. Chem. 276, 993-998.
  6. Rahdar M. et al. 2009. A phosphorylation-dependent intramolecular interaction regulates the membrane association and activity of the tumor suppressor PTEN. Proc. Natl. Acad. Sci. USA. 106, 480-485.
  7. Yim E.K. et al. 2009. Rak functions as a tumor suppressor by regulating PTEN protein stability and function. Cancer Cell. 15, 304-314.
  8. Maddika S. et al. 2011. WWP2 is an E3 ubiquitin ligase for PTEN. Nat. Cell Biol. 13, 728-733.
  9. Li Z. et al. 2005. Regulation of PTEN by Rho small GTPases. Nat. Cell Biol. 7, 399-404.
  10. Trotman L.C. et al. 2007. Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell. 128, 141-156.
  11. González-Santamaría J. et al. 2012. Regulation of the tumor suppressor PTEN by SUMO. Cell Death Dis. 3, e393.
  12. Huang J. et al. 2012. SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane. Nat. Commun. 3, 911.
  13. Bassi C. et al. 2013. Nuclear PTEN controls DNA repair and sensitivity to genotoxic stress. Science. 341, 395-399.
  14. Okumura K. et al. 2006. PCAF modulates PTEN activity. J. Biol. Chem. 281, 26562-26568.
  15. Ikenoue T. et al. 2008. PTEN acetylation modulates its interaction with PDZ domain. Cancer Res. 68, 6908-6912.
  16. Cho S.H. et al. 2004. Redox regulation of PTEN and protein tyrosine phosphatases in H2O2-mediated cell signaling. FEBS Lett. 560, 7-13.
  17. Cao J. et al. 2009. Prdx1 inhibits tumorigenesis via regulating PTEN/AKT activity. EMBO J. 28, 1505-1517.
  18. Horita H. et al. 2017. Identifying regulatory posttranslational modifications of PD-L1: A focus on monoubiquitinaton. Neoplasia. 19, 346-353.
  19. Xu W. et al. 2014. Posttranslational regulation of phosphatase and tensin homolog (PTEN) and its functional impact on cancer behaviors. Drug Des. Devel. Ther. 8, 1745-1751.

 

Shi Y. et al. 2012. PTEN at a glance. J. Cell Sci. 125, 4687–4692.
Lee J.O. et al. 1999. Crystal structure of the PTEN tumor suppressor: Implications for its phosphoinositide phosphatase activity and membrane association. Cell. 99, 323-324.
Song M.S. et al. 2012. The functions and regulation of the PTEN tumor suppressor. Nat. Rev. Mol. Cell Biol. 13, 283-296.  
Al-Khouri A.M. et al. 2005. Cooperative phosphorylation of the tumor suppressor phosphatase tensin homologye (PTEN) by casein kinases and glycogen synthase kinase 3beta. J. Biol. Chem. 280, 35195-35202.
Torres J. and Pulido R. 2001. The tumor suppresspr PTEN is phosphorylated by the protein kinase CK2 at its C terminus. Implications for PTEN stability to proteasome-mediated degradation. J. Biol. Chem. 276, 993-998.
Rahdar M. et al. 2009. A phosphorylation-dependent intramolecular interaction regulates the membrane association and activity of the tumor suppressor PTEN. Proc. Natl. Acad. Sci. USA. 106, 480-485.
Yim E.K. et al. 2009. Rak functions as a tumor suppressor by regulating PTEN protein stability and function. Cancer Cell. 15, 304-314.
Maddika S. et al. 2011. WWP2 is an E3 ubiquitin ligase for PTEN. Nat. Cell Biol. 13, 728-733.
Li Z. et al. 2005. Regulation of PTEN by Rho small GTPases. Nat. Cell Biol. 7, 399-404.
Trotman L.C. et al. 2007. Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell. 128, 141-156.
González-Santamaría J. et al. 2012. Regulation of the tumor suppressor PTEN by SUMO. Cell Death Dis. 3, e393.
Huang J. et al. 2012. SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane. Nat. Commun. 3, 911.
Bassi C. et al. 2013. Nuclear PTEN controls DNA repair and sensitivity to genotoxic stress. Science. 341, 395-399.
Okumura K. et al. 2006. PCAF modulates PTEN activity. J. Biol. Chem. 281, 26562-26568.
Ikenoue T. et al. 2008. PTEN acetylation modulates its interaction with PDZ domain. Cancer Res. 68, 6908-6912.
Cho S.H. et al. 2004. Redox regulation of PTEN and protein tyrosine phosphatases in H2O2-mediated cell signaling. FEBS Lett. 560, 7-13.
Cao J. et al. 2009. Prdx1 inhibits tumorigenesis via regulating PTEN/AKT activity. EMBO J. 28, 1505-1517.
Horita H. et al. 2017. Identifying regulatory posttranslational modifications of PD-L1: A focus on monoubiquitinaton. Neoplasia. 19, 346-353.
Xu W. et al. 2014. Posttranslational regulation of phosphatase and tensin homolog (PTEN) and its functional impact on cancer behaviors. Drug Des. Devel. Ther. 8, 1745-1751.