June Kinoshita, with Peter Davies, led this live discussion on 3 August 1999. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.

Paper under discussion: Lu, Pei-Jung, Wulf, Gerburg, Zhou, Xiao Zhen, Davies, Peter, and Lu, Kun Ping. The propyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein. Nature 399, 784-788. 1999 Jun 24. Abstract

View Transcript of Live Discussion — Posted 5 September 1999

Background Text
By Peter Nelson

Nature is under no obligation to be simple. The complexity of reality is underscored in the intriguing new paper by Pei-Jung Lu and colleagues regarding a role for mitosis-related Pin1 protein in tau protein biochemistry (ref. 7). The paper argues for an interaction that is important in both normal and Alzheimer's disease brain. Moreover, it involves a (relatively) novel paradigm of protein-protein interaction. This finding is of interest in many fields of biosciences, but in an applied way could be most germane to neurological conditions including Alzheimer's disease
The purpose of this mini-review is to provide some background into the different fields of biosciences that are pertinent to an understanding of the abovementioned paper; to discuss the paper itself; and to suggest some questions and ideas for discussion on this web page and the accompanying live chat.
When diverse scientific fields are brought together in a novel advancement, sparks fly, and scientists rush to the library to get background papers. The paper by Lu and colleagues involves the biochemistry of the cell cycle and related proteins; tau proteins, in normal and Alzheimer states; and Pin1 and the paradigm of phosphorylation-dependent prolyl isomerases. These separate "players" had given some hints of interacting (e.g., as described below), but never before in this direct manner.
The cell cycle refers to the process by which cells and their genes regulate replication, division, differentiation and apoptosis. There is a cyclic progression:

G0<-->G1(first "gap")-->S(DNA synth)--->G2(second gap)--->M(mitosis)--->back to G1

This basic schema has been appreciated for years. In the past decade, however, an almost staggering amount of information has been added to describe how this cycle is regulated in healthy and diseased tissue. No doubt, more will come.

Cell Cycle Proteins in Alzheimer's Disease
The role of cell cycle proteins in Alzheimer's disease has been both suggestive and puzzling. For one thing, Alzheimer's disease presents clinically long after the cell cycle in neurons "should" be frozen (in G0). Yet cell cycle proteins have been detected via immunohistochemistry along with Alzheimer's pathology in neurons. For example, the following proteins have been detected up-regulated in Alzheimer's-affected neurons relative to controls: Ki-67 (an indicator of being out of the G0 state), Cyclin D (at G1/S interface), and Cyclin B1 (G2/M) (refs. 1,9,10). Other hints of mitotic activity involve the familial Alzheimer's-related gene, presenilin (ref. 6). If these neurons are not dividing, what are they doing? Presumably, there is a link between how cells divide and differentiate on the one hand, and how they cope with injury on the other. In Alzheimer's brain, there is apparently an up-regulation of pathways reminiscent of cycling cells. It remains to be seen if this response contributes to the health or further injury of a given nerve cell, and if it exacerbates the overall disease process.
Many of the cell cycle proteins are kinases, which attach phosphate moieties to proteins. In 1996, Lu and colleagues described a new paradigm for how protein "modules" (ref.6), including so-called WW domains, can be employed to attach one protein to another in a phosphorylation- dependent manner (downstream of kinase action). This paradigm had been foreshadowed by the manner in which SH2 domains facilitate binding when one protein contains a phosphorylated tyrosine residue. However, WW domains involve phosphorylated serines or threonines, and the WW domain interaction causes an "isomerase" bending of the affected protein molecule. Since the WW domains are a module that can attach to any protein, they are quite versatile.

Protein 1----------Phosphorylated Serine/Threonine

{protein-protein binding}

WW domain-mediated interaction----------------------Protein 2

{Change in Protein 2 function}

Pin1 (peptidyl-prolyl isomerase nucleoprotein) is an example of a versatile protein that contains a WW domain (key references include refs. 2,3,4,6,7,8,12,13,14,15). Most of the known substrates to which Pin1 binds through its WW domain are mitotic phosphoproteins. For example, Pin1 interacts with phosphorylated kinases (e.g., myt1, plk1), phosphatases (e.g. Cdc27), proteases (e.g. Nedd4), and the GTP binding protein Rab4. An important-seeming interaction involves Cdc25 and plx1, upstream regulators of Cdc2/cyclin B, in a manner that renders Pin1 an essential regulator of the cell cycle (too much Pin1 leads to cell cycle arrest in G2; depletion of Pin1 causes arrest of mitosis[ref.5]).

Summary of Paper Under Discussion
In the paper being presently discussed, Lu et al. provide data consistent with the hypothesis that that Pin1 and tau can interact with high specificity (ref.7). This begs the question: is tau protein a "mitotic phosphoprotein"? The prevailing hypotheses pertaining to tau protein focus on its role in tubulin binding and the subsequent seeding/bundling of microtubules. Through the effect on microtubules, tau is thought to play a role in the elaboration of neuronal processes (mainly the axon), developmental axon plasticity, and the maintenance of cell shape. However, some previous data support a connection between tau protein and cell cycle biochemistry. Tau protein has been shown to be:

• minimally phosphorylated in neuronal cell lines in culture during interphase of cell division
• highly phosphorylated in the same cell lines during mitosis (ref.11)
• phosphorylated by cell cycle kinases
• present in the nuclei of cells under certain circumstances
• phosphorylated in neurofibrillary tangles of Alzheimer's disease in the same (as well as other) residues as during mitosis

These and other data imply a role for tau in cell cycle biochemistry (moreover, there is a lot of data to suggest that tau proteins may play other important roles in neuronal cell biology; remember, nature is under no obligation to be simple!). Pei-Jung Lu and colleagues have greatly extended the work connecting tau proteins and the cell cycle by the present study involving Pin1 and tau (ref. 7). Their findings include:

• Tau proteins phosphorylated by mitotic Xenopus extracts (but not by interphase extracts) bind to GST-bound Pin1 in a pull-down assay
• Likewise, AD tau but not normal tau binds GST-Pin1
• Pin-1 binds to AD neurofibrillary tangles in situ.
• Pin-1 is immunohistochemically found in normal neuronal nuclei
• Pin-1 binds with high specificity to a single phosphorylated threonine residue on tau (pT231)
• Tau that has been phosphorylated by Cdc2 binds to GST-Pin1 via a WW domain at pT231
• Cdc2-phosphorylated tau has reduced microtubule-forming capabilities
• Pin-1 binding of Cdc2-phosphorylated tau re-enables microtubule forming capabilities

Kinases are differentially depleted in Alzheimer brain neurons; whereas GSK-3-beta levels are relatively unchanged, and Cdc2 levels are increased, Pin-1 levels are decreased by approximately a factor of 5, leading the authors to posit that "sequestration of Pin-1 in PHFs depletes soluble Pin-1, which itself might also have a deleterious effect."

Some Discussion Topics
Whether all of the findings in this study will "stand up" to future critical analyses is unknown, obviously. What does seem robust is the interaction between Pin1 and phosphorylated tau at the pT231 residue. T231 resides in an evolutionarily well-conserved area of tau protein N-terminal to the microtubule-binding repeats (there are some mutations among mammalian species in residues between T231 residue and the binding repeats) (ref.11).
This study, as good studies do, raises novel questions. These include:

• What is the pharmacology of the interaction between Pin1 and tau (can it be stimulated to improve the function of tau in Alzheimer's disease and other disorders with tangles?)?
• Does this pathway have a direct role in cell death?
• How important a role does tau have in neuronal mitoses?
• Why is mitosis relevant at all in the context of mature neurons?
• Does Pin1 directly "seed" PHFs?
• What is the biochemistry of tau proteins in the nucleus, if any?

In addition to the direct relevance to Alzheimer's disease, the interaction of Pin1 and tau is titillating to researchers interested in tau molecule per se. Here is a novel mechanism that allows phosphorylated tau protein to be added on, almost like a Lego piece, to any module-containing protein. The implications are fantastic, almost limitless, to the applications that can now be performed by tau. It could help to explain the many diverse roles that have been suggested for tau (e.g. interactions with vesicles and ribosomes), as it may also help to explain why tau is so finicky an antigen in situ.
It may seem complicated and daunting to think of now, but, come to think of it, things tend to appear a bit simpler, in retrospect.

References
1. Busser J, Geldmacher DS, Herrup K. Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer's disease brain. J.Neurosci. 1998 Apr 15;18:2801-2807. Abstract.

2. Campbell HD, Webb GC, Fountain S, Young IG. The human PIN1 peptidyl-prolyl cis/trans isomerase gene maps to human chromosome 19p13 and the closely related PIN1L gene to 1p31. Genomics 1997 Sep 1;44:157-162. Abstract.

3. Crenshaw DG, Yang J, Means AR, Kornbluth S. The mitotic peptidyl-prolyl isomerase, Pin1, interacts with Cdc25 and Plx1. EMBO J. 1998 Aug 10;17:1315-1327. Abstract.

4. Gothel SF, Marahiel MA. Peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts. Cell Mol.Life Sci. 1999 Mar;55:423-436. Abstract.

5. Li,J.; Xu,M.; Zhou,H.; Ma,J.; Potter,H. Alzheimer presenilins in the nuclear membrane, interphase kinetochores, and centrosomes suggest a role in chromosome segregation. Cell 1997: 90:917-927. Abstract.

6. Lu KP, Hanes SD, Hunter T. A human peptidyl-prolyl isomerase essential for regulation of mitosis. Nature 1996 Apr 11;380:544-547. Abstract.

7. Lu, Pei-Jung, Wulf, Gerburg, Zhou, Xiao Zhen, Davies, Peter, and and Lu, Kun Ping. The propyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein. Nature 399, 784-788. 1999 Jun 24. Abstract.

8. Lu PJ, Zhou XZ, Shen M, Lu KP. Function of WW domains as phosphoserine- or phosphothreonine-binding modules. Science 1999 Feb 26;283:1325-1328. Abstract.

9. Nagy Z, Esiri MM, Cato AM, Smith AD. Cell cycle markers in the hippocampus in Alzheimer's disease. Acta Neuropathol.(Berl.) 1997 Jul;94:6-15. Abstract.

10. Nagy, Z, Esiri MM, Smith AD. Expression of cell division markers in the hippocampus in Alzheimer's disease and other neurodegenerative conditions. Acta Neuropathol. (Berl.) 1997 Mar; 93: 294-300. Abstract.

11. Nelson, P.T., Stefansson, K., Gulcher, J., Saper, C.B. Molecular evolution of Tau protein: implications for Alzheimer's disease. J. Neurochem. 1996 Oct; 67:1622-1632. Abstract.

12. Preuss U, Doring F, Illenberger S, Mandelkow EM. Cell cycle-dependent phosphorylation and microtubule binding of tau protein stably transfected into Chinese hamster ovary cells. Mol.Biol.Cell 1995 Oct;6:1397-1410. Abstract.

13. Ranganathan R, Lu KP, Hunter T, Noel JP. Structural and functional analysis of the mitotic rotamase Pin1 suggests substrate recognition is phosphorylation dependent. Cell 1997 Jun 13; 89:875-886. Abstract.

14. Shen M, Stukenberg PT, Kirschner MW, Lu KP. The essential mitotic peptidyl-prolyl isomerase Pin1 binds and regulates mitosis-specific phosphoproteins. Genes Dev. 1998 Mar 1;12:706-720. Abstract.

15. Yaffe MB, Schutkowski M, Shen M, et al. Sequence-specific and phosphorylation-dependent proline isomerization: a potential mitotic regulatory mechanism. Science 1997 Dec 12;278:1957-1960. Abstract.

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