DEF6: The ins and outs

DEF6 is a curious molecule. Whilst it is crucial for the proper function of our adaptive immune system, it was only isolated in 2003 and still very little is known about it. I am currently in the midst of my fourth year biology project investigating this protein and thought I would share some details of what I’m up to!

 

Before I get all science-y about what I am doing, let me first explain what DEF6 actually is. It is a protein found primarily in T-cells – a type of cell found in the adaptive immune system that differentiates and activates an immune response when it comes into contact with a pathogen. DEF6 acts as a guanine nucleotide exchange factor (GEF) in these cells and is composed of four domains; at one end is an EF-hand domain which is next to a DEF6-SWAP70 Homology (DSH) domain. Next to this is a Pleckstrin Homology (PH) domain and then ends with a Dbl homology-like (DHL) – the picture below sums all this up! As DEF6 is not hugely researched, the full functions of these domains are not entirely clear, with the DSH currently having an unknown effect on the protein. EF-hands have previously been shown to be involved in calcium ion (Ca2+) binding, whilst the PH and DHL determine the function of DEF6. These domains are characteristic of Rho-family (a specific subset) GEFs but only in DEF6 (and its close homologue SWAP70) are these domains in reverse order.

 

Screen Shot 2017-03-09 at 16.22.14

The domain sequence in DEF6

 

You’re probably wondering what actually is a guanine nucleotide exchange factor? GEFs work in conjunction with other proteins called GTPases. These are proteins which are normally bound to a molecule called guanosine diphosphate (GDP) in an inactive state. GDP can be swapped for guanosine triphosphate (GTP) – GDP with an extra phosphate attached – to make the GTPase change to an active state where it can then bind to other proteins and activate them in turn. A GEF comes into this by helping the GTPase bind to a GTP molecule instead of a GDP. After a while, the GTPase breaks down the bound GTP to GDP and once again becomes inactive, detaching from the protein it was previously activating. This last step can be carried out faster with the help of a GTPase activating protein (GAP) which reduces the time the GTPase needs to break down the GTP. The picture just below sums up this process.

 

Screen Shot 2017-03-09 at 16.55.05

The mechanism of alternating GTPase activity.

 

As DEF6 can’t always be doing its thing 24/7, it needs to be regulated in some way. Enter the phosphorylating enzymes! DEF6 is able to be switched on and off by respectively adding or removing phosphate molecules to specific sites in its sequence, termed phosphorylation. DEF6 has been found to be phosphorylated by two enzymes; the tyrosine kinases Lck and Itk. As the name suggests these enzymes add phosphates explicitly to the amino acid tyrosine in the protein’s sequence, more specifically Lck phosphorylates two tyrosines in the DSH domain, whilst Itk phosphorylates once in the DSH and the PH domain. These additions of phosphate molecules cause the DEF6 to switch from inactive to active.

 

In the context of T-cells, DEF6 becomes phosphorylated once the T-cell ‘recognises’ and binds to a foreign pathogen in the blood causing a cascade of biological signalling within the cell, part of which is DEF6’s phosphorylation by Lck and Itk. DEF6 is now active and able to function as a GEF for the Rho-family GTPases Cdc42 and Rac-1. These two GTPases can induce the differentiation and proliferation of T-cells via molecules known as transcription factors – a protein that causes the transcription and translation of certain genes. If that wasn’t confusing enough, Cdc42/Rac-1 can also upregulate the construction of actin filament which bind the T-cell to the pathogen to ensure the signalling is prolonged for an adequate immune response.

 

My project specifically focuses on the findings that additional phosphorylations of threonine and serine residues in the DHL domain have been found – not only in humans – but also in rats and mice at the same sites. It is my aim to investigate a handful of these DHL domain phosphorylations, by mutating the threonines and serines to amino acids that mimic phosphorylation or non-phosphorylated sites in DEF6 to see how the protein behaves when continuously active/inactive, respectively. This mutated version of DEF6 will then be inserted into a specific cell line called COS-7 cells which are cells derived from the African green monkey’s kidney tissue, and are able to produce actin filaments (a type of structural filament in cells that acts like their skeleton) similar to when DEF6 is active in T-cells.

 

Hopefully by the end, my project will reveal some new information on the function and effect of phosphorylation on DEF6. Deficiency in DEF6 has been shown to quickly exhibit symptoms of autoimmune diseases and can lead to illnesses such as rheumatoid arthritis and lupus. These two diseases can severely impact upon the lives and welfare of those affected, so any new light shed upon DEF6 may help towards the treatment and diagnosis of these diseases.

 

Here is some further reading if you would like to read more:

DEF6 phosphorylated by Itk: http://www.jbc.org/content/287/37/31073.full.pdf

DEF6  phosphorylated by Lck: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2801603/

Rho-family GTPases: http://www.biochemsoctrans.org/content/40/6/1378

DEF6 as a GEF for Cdc42 and Rac-1: http://www.jbc.org/content/278/44/43541.long

 

Title image: A GFP-tagged DEF6 transfected COS-7 cell taken by myself

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