Study of the effect of ATPase activity of 14-3-3 γ and ε isoforms on desmosome assembly and cell cycle control

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4. Project Title

Study of the effect of ATPase activity of 14-3-3 γ and ε isoforms on desmosome assembly and cell cycle control

5. Project Summary

14-3-3 is a family of highly conserved proteins that are known to play a role in the maintenance of signalling pathways and in the regulation of cellular processes including DNA repair, cell cycle checkpoint control, cellular differentiation, motility, adhesion, apoptosis and senescence. The seven isoforms of these proteins act either through the sequestration of proteins or by inducing conformational changes in their ligands. However it has also been suspected that the 14-3-3 proteins may themselves possess some enzymatic activity that plays a role in their functioning.

In this regard, a recent study has demonstrated for the first time that 14-3-3 ζ possesses ATPase activity. However, the conservation of this function among the related isoforms of the protein as well as its functional and biological relevance has yet to be explored.

This project aims to study the ATPase activity of the 14-3-3 γ and ε isoforms. Further, it is hypothesised that since these isoforms play a major role in cell cycle checkpoint regulation and desmosome assembly, such ATPase activity could have an effect on these functions.

Initial studies carried out by the lab indicate that 14-3-3 γ and ε appear to have ATPase activity that is comparable to that of 14-3-3 ζ. Therefore our hypothesis can be tested by generating gain-of-function and loss-0f-function mutants of the two isoforms, quantifying their ATPase activity and then transfecting mammalian cells with the mutant gene and studying the effect that this has on the cell cycle and its regulation as well as the phenotypic characteristics of desmosome assembly.

This research project has a projected budget of ₹ 13,00,000 and is expected to require a period of 4-6 months for completion.

This study is particularly relevant because impairments in the processes of cell cycle regulation and cell adhesion are directly linked with the onset and progression of carcinogenesis and malignancy. Therefore an elucidation of the properties and mechanisms of action of the 14-3-3 family of proteins is an essential step towards better understanding disease progression as well as the early detection, diagnosis and ultimately the cure of cancer.

6. Details of the project

6.a. Introduction

6.a.i. Origin of the Proposal

14-3-3 refers to a family of highly conserved acidic, dimeric proteins that are present ubiquitously in eukaryotes.1 They were first discovered in brain tissue where they were found to be among the most abundant proteins; and subsequently it was discovered that were present in small amounts in almost all tissues.2 In animals seven different 14-3-3 isoforms have been identified to date - β, γ, ε, ζ, σ, η and τ.3

These proteins play a crucial role in the maintenance of multiple signalling pathways and in the regulation of cellular processes like DNA repair, cell proliferation and cell cycle checkpoint control, gene transcription, cellular differentiation, motility, adhesion, apoptosis and senescence.4 The activity of these proteins therefore involves various binding partners and a highly complex signalling network.4

14-3-3 proteins are known to bind primarily to specific phosphorylated serine/threonine- containing sequences within their binding partners.5 However, it has been found that even though this family of proteins is highly conserved, the different isoforms do not do not share the same specificity and do not interact equivalently with their ligands.4 As a result, changes or mutations in specific isoforms can affect different aspect of cell signalling in complex ways.

The mechanism of action of the proteins in this family is thought to involve both the sequestration of proteins within particular cellular compartments as well as the modification of the enzymatic activity of its ligands through induction of conformational changes.3,6,7 However it is also suspected that the 14-3-3 proteins may themselves possess some enzymatic activity that plays a role in their functioning. An indication of such activity was provided when an ATP/ADP exchange function shown to be possessed by the 14-3-3 τ isoform.8 Nevertheless, the role of this enzymatic activity remained controversial in the absence of conclusive proof.

6.a.ii.a. Rationale of the study

A recent study conducted on 14-3-3 ζ has demonstrated for the first time that the protein possesses ATPase activity.9 The study also identified individual residues that play an important role in this ATPase activity by generating mutants and screening them for gain- or loss- of function.9

The gain of function mutant in this study was generated by mutating the Aspartate at position 124 to Alanine (D124A); while the loss of function mutant was generated by mutating the Arginine at position 55 to Alanine (R55A). What is interesting about both these mutants is that in the case of 14-3-3 ζ, both the mutated residues - D124 as well as R55, lie within the protein’s phosphopeptide binding pocket.9 Therefore, the mutation of these residues, apart from regulating the protein’s ATPase activity, could also affect the binding of its phosphopeptide ligands, thus mediating a downstream effect.4,10,11,12

Such functional and biological relevance of this ATPase activity has however not yet been explored. Additionally, since 14-3-3 represents a family of highly conserved proteins, the discovery of ATPase activity in one isoform opens up several new avenues to study similar activity in the other 14-3-3 isoforms as well.

This project aims to study the ATPase activity of the 14-3-3 γ and ε isoforms. Further, since these isoforms play a major role in cell cycle checkpoint regulation and desmosome assembly, we wish to study whether the alteration of ATPase activity has any effect on these processes.

6.a.ii.b. Hypothesis

If the ATP hydrolysing activity of 14-3-3 γ and ε isoforms plays a role in its binding to other proteins or its downstream signalling, then mutations in this activity could have implications on the assembly and localization of desmosomes, as well as on the regulation of the cell cycle.

6.a.ii.c. Key questions

1. Do 14-3-3 γ and ε isoforms possess ATP hydrolysing activity?

2. Can the mutation of individual amino acids bring about a gain or loss of this activity?

3. Does this ATP hydrolysing activity have any implications on the known functions of the γ and ε isoforms?

6.a.iii.a. Current status of Research & Development (National and International)

14-3-3 proteins

Owing to the association of 14-3-3 proteins with a wide array of essential cellular functions, this protein has been at the heart of the attention of a number of research groups. Early studies focussed on the structure, composition and binding properties of these proteins. Among the most important of these is the study carried out by Yaffe et. al. in 1997, which identified two optimal binding motifs for the 14-3-3 family: RSXpSXP and RX(Y/F)XpSXP (where X represents any amino acid except cysteine).13 Later in 2005, Wilker et. al. also demonstrated that all the 14-3-3 proteins except 14-3-3 σ form homo and heterodimers.14

More recent studies have been focussed on the biological functions and mechanism of action of the 14-3-3 proteins and their role in development of cancer. The aberrant expression of 14-3-3 isoforms has been found to be associated with several human cancers. Numerous studies have been able to demonstrate that 14-3-3σ acts as a tumor-suppressor gene and that its inactivation is one of the crucial steps involved in tumorigenesis.4

14-3-3 proteins and the cell cycle

14-3-3 is known to play a major role in cell cycle progression and regulation through its association with target proteins.15 One of the known targets of 14-3-3 γ and ε is the phosphatase Cdc25C, which plays a role in the activation of the CDK1/CyclinB1 complex which in turn causes the progression of the cell cycle from the G2 to M phase and drives the cell through mitosis.16 During the interphase, 14-3-3 proteins bind to Cdc25C and sequester it in the cell cytoplasm, thus in effect inactivating it and leading to the cell cycle getting arrested in the G2 phase.16 14-3-3 ε and β also inhibit Cdc25B by a similar mechanism.17,18

Additionally, 14-3-3 proteins also control the cell cycle by activating the enzyme wee1. Wee1 is a kinase, which in its activated form inhibits CDC2 from being phosphorylated and thus blocks cell cycle progression.19 Thus 14-3-3 proteins play an active and essential role in regulation of the cell cycle, and the loss of 14-3-3 ε and γ leads to premature entry into mitosis due to overriding of the checkpoint function.19

14-3-3 proteins and the cell adhesion

14-3-3 proteins are also known to play a role in controlling cellular adhesion and migration through the regulation of integrins and desmosomes. 14-3-3β is known to interact with the cytoplasmic fragment of the integrin β1 to stimulate cell spreading.20 In a study conducted by Han et. al., the overexpression of 14-3-3β was thus able to diminish cell adhesion and bring about an increase in cell migration.21

In contrast, the interaction of 14-3-3γ with desmosomal proteins is said to promote the assembly of adhesion junctions. A study conducted in 2014 by Sehgal et. al. demonstrated that the loss of 14-3-3γ led to a decrease in cell–cell adhesion and defective localization of plakoglobin and other desmosomal proteins to the cell border. This study conclusively demonstrated that 14-3-3γ is required for normal desmosome assembly and function.22

6.a.iii.b. Preliminary work done by the lab

Initial studies carried out by the lab indicate that 14-3-3 γ and ε appear to have ATPase activity that is comparable to that of 14-3-3 ζ. Additionally, mutation of the conserved residues corresponding to 14-3-3 ζ R55 and D124 are also appear to be able to produce gain and loss of function variants of the protein with respect to ATPase activity. However no work has been done on the biological implications of these mutations.

6.a.iv. Relevance and expected outcome

This study is expected to provide us with greater insight into any ATPase activity that the 14-3-3 γ and ε isoforms may possess, as well as its implications on their known functions. A mutation in the 14-3-3γ or ε proteins that either enhances or eradicates such ATPase activity may thus cause aberrant phenotypes with respect to desmosome assembly and cell cycle regulation.

This study is particularly relevant because any impairment in these processes may be directly linked with the onset and progression of carcinogenesis and malignancy. Therefore an elucidation of the properties and mechanisms of action of the 14-3-3 family of proteins is an essential step towards better understanding disease progression as well as the early detection, diagnosis and ultimately the cure of cancer.

6.b. Objectives

  1. To generate and clone genes coding for gain of function and loss of function 14-3-3 γ and ε mutants
  2. To study the ATPase activity of the 14-3-3 γ and ε mutants
  3. To study the effect of ATPase activity of 14-3-3 proteins on desmosomes and their assembly
  4. To study the effect of ATPase activity of 14-3-3 proteins on cell cycle regulation

6.c. Work Plan

To generate and clone genes coding for gain of function and loss of function 14-3-3 γ and ε mutants Time period: 3-4 weeks

Specific aim 1.1: To generate 14-3-3 γ and ε gene mutants

This will be done by Site Directed Mutagenesis by PCR, using γ and ε isoform-specific primers; and plasmids bearing the corresponding 14-3-3 genes as the templates. These primers will incorporate mutations that correspond to the R55A and D124A mutations of 14-3-3 ζ.

Specific aim 1.2: To generate bacterial clones carrying the mutant genes

The vectors bearing the mutant 14-3-3 genes will be used to transform competent E.coli DH5α cells. Cells will be screened using media plates containing antibiotics corresponding to the resistance marker carried by the vector, and the positive clones will then be maintained individually.

Specific aim 1.3: Screening of clones

Clones appearing to be positive for the selectable marker will be screened individually by extracting plasmid DNA from the isolated cultures and sequencing it to check for the mutation of interest as well as any PCR introduced errors.

To study the ATPase activity of the 14-3-3 γ and ε mutants

Time period: 3-4 weeks

Specific aim 2.1: Cloning the mutant genes into expression vectors

The generated mutant genes will be excised from the existing plasmid and cloned into a bacterial expression vector such as pGEX-4T-1. This expression vector bearing the insert will then be used to transform competent cells; of which the positive clones will be isolated and screened.

Specific aim 2.2: Expression and purification of the mutant 14-3-3 proteins

The 14-3-3 mutant gene will be expressed in the host cells in response to IPTG induction. The expressed protein will then be purified from the cell lysate based on either a GST- or a His- tag, using an affinity chromatography column. The tag will then be cleaved from the peptide to yield a pure protein preparation.

Specific aim 2.3: Quantification of ATPase activity

The ATP hydrolysing activity of the purified protein will then be estimated by either a colorimetric assay using malachite green, a radiolabeled assay using (γ-32P) ATP or an ADP-GloTM Max assay (Promega); with the un-mutated wild type 14-3-3 protein as the control.

To study the effect of ATPase activity of 14-3-3 proteins on desmosomes and their assembly

Time period: 4-5 weeks

Specific aim 3.1: To generate stable clones of mammalian cells expressing the mutant 14-3-3 proteins

Mammalian cell lines will be cultured in vitro and transfected with vectors containing mutant 14-3-3 genes. The transfected cells will be screened for stable clones and these clones will be subcultured and further used study the effect of 14-3-3 proteins on desmosomes and the cell cycle.

Specific aim 3.2: To study the interaction of mutant 14-3-3 ATPase mutants with desmosomal proteins in vitro

The interaction of the 14-3-3 mutants with desmosomal proteins can be studied by using the lysate of transfected mammalian cells and applying techniques such as immunoprecipitation and Western blotting. The un-mutated wild type 14-3-3 protein will be used as the control.

Specific aim 3.3: To study the effect of mutant 14-3-3 proteins on the assembly and cellular localization of desmosomes

Whole cells transfected with the mutant 14-3-3 proteins can also be studied by Immunofluorescence, using labelled antibodies in order to examine whether the desmosomes are getting localized to the cell border as expected, or not.

To study the effect of ATPase activity of 14-3-3 proteins on cell cycle regulation

Time period: 4-5 weeks

Specific aim 4.1: To study the effect of mutant 14-3-3 proteins on mitosis

In order to see whether the ATPase activity of 14-3-3 plays any role in its regulation of the cell cycle and mitosis, the cell cycle progression of transfected mammalian cells will be monitored and the mitotic index will be calculated.

Specific aim 4.2: To study the effect of mutant 14-3-3 proteins on cdc25c function

The wild type 14-3-3 γ and ε are able to bind to the cdc25c protein in vivo and inhibit its ability to induce premature chromatin condensation (PCC). To check whether the mutant proteins are functionally equivalent, transfected mammalian cells will be studied in order to determine the extent to which PCC induction is inhibited in them, as compared to the wild type protein-bearing cells.

6.d. References

7. Duration of the project

4 months (1st December 2014 to 31st March 2015)

8. Budget

Heads

Particulars

Amount (₹)

Equipment

Thermocycler, Horizontal Electrophoresis Apparatus, Micropipettes,

7,00,000

Consumables

Microcentrifuge tubes, Micropipette tips, petriplates, PCR tubes, 96 well plates

1,00,000

Cell lines and cell strains

E. coli DH5α, E. coli BL-21, Human Colon Carcinoma cell line HCT-116

15,000

Biological reagents

Oligonucleotide primers, Polymerase enzymes, dNTP’s Restriction endonuclease enzymes, Fluorescence labelled antibodies, Trypsin, RNase, Proteinase K

3,00,000

Chemical reagents

SDS, Tris Base, EDTA, EtBr, Agarose, Caesium Chloride

85,000

Services

DNA sequencing, Fluorescence confocal microscopy

50,000

Overheads

Electricity, Water (MilliQ)

50,000

TOTAL

13,00,000

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