Multiple Sequence Alignment

We have performed a first multiple sequence alignment of the four human isoforms, followed by a multiple sequence alignment of the human isoform a (which is the most abundant isoform in humans), the LAT homologue in rat, the LAT homologue in mouse and the LAT homologue in cow.

This was done using the Clustal W tool provided by the Swiss Institute of Bioinformatics , followed by BOXSHADE 3.21 which is a printing and shading of multiple alignment files.

According to the predicted linear model presented here, we have indicated the possible domains interacting with PLC- γ1 and with GRB2, GRAP2 and PIK3R1. We have also indicated the extracellular topological domain and transmembrane region of the human isoforms

Multi sequence alignment of the human LAT isoforms

From analysing four isoforms of LAT our MSA shows large sections of the protein are conserved across all forms (shown by the amino acids highlighted in black). The amino acids that have been identified as key sites for signal transduction are preserved in all four isoforms indicating that they are able to carry out the same functions. Isoform A is the most common form of LAT protein found so for the next multiple sequence alignment we have just compared to this.

Multi sequence alignment of the LAT homologues

From this MSA you can see there are many sections of the protein that are conserved across all four species, shown by the amino acids highlighted black. There are also individual amino acids highlighted grey showing that the sequence contains many amino acids with similar characteristics/properties to those in the same position on the different forms of LAT. This is important for binding to ensure the same regions always have distinct properties so can be recognised by their target substrates, for example residues with the binding site could be hydrophobic, negatively charged etc..

Using the predicted linear model we have produced, we have indicated the possible domains interacting with PLCγ1 , GRB2, GRAP2 and PIK3R1. These domains are conserved across all the alignments showing all the different forms of LAT bind to their target in the same regions. GRB2, GRAP2 and PIK3R1 all bind via their SH2 domain to the same region on the LAT protein [1]; this shows these proteins under certain conditions all recognise the same consensus sequence. SH2 domains recognise and bind to tyrosine phosphorylated sites. Looking at the highlighted interaction domains the tyrosine residue is always followed by Valine and Asparagine, hydrophobic and polar residues. We know the SH2 domain on GRB2 preferably binds to Y-X-N-X [2], where X is a hydrophobic residue (eg.Valine), which fits perfectly with all these alignments.
PLC γ1 also interacts with phosphorylated tyrosine residues [1] by cleaving the phospholipid. From the interaction domain for PLC γ1 the tyrosine residue this time is followed by Valine and Leucine residues, which are both hydrophobic.

The first four residues of LAT isoform a are the extracellular domain which are hydrophobic or negatively charged this is followed by the membrane spanning region (23 residues); these are mainly hydrophobic as well.

The differences between any of the different forms of LAT could be due to alternative splicing; rearrangement of RNA exons produced during transcription of LAT gene. Both the LAT form found in mice and rats show huge sections that are missing compared to the Human isoform a and LAT found in cows, this could be due to a deletion due to the information coded not being required in the rat or mouse. A – indicates missing parts of the gene compared to other alignments.

References

[1]. Wange R.L, (2000). “LAT, the linker for activation of T cells: a bridge between T cell-specific and general signaling pathways”. Science's STKE : signal transduction knowledge environment, Vol.2000(63), p.re1.

[2]. Nioche P, (2002). “Crystal structures of the SH2 domain of grb2: highlight on the binding of a new high-affinity inhibitor”. Journal of Molecular Biology, Vol.315(5), p.1167-1177.


		*Multiple Sequence Alignment* We have performed a first multiple sequence alignment of the four human isoforms, followed by a multiple sequence alignment of the human isoform a (which is the most abundant isoform in humans), the LAT homologue in rat, the LAT homologue in mouse and the LAT homologue in cow. This was done using the Clustal W tool provided by the Swiss Institute of Bioinformatics , followed by BOXSHADE 3.21 which is a printing and shading of multiple alignment files. Colour code: Black: conserved residues Grey: conservative mutations White: divergence According to the predicted linear model presented here, we have indicated the possible domains interacting with PLC- γ1 and with GRB2, GRAP2 and PIK3R1. We have also indicated the extracellular topological domain and transmembrane region of the human isoforms MULTIPLE SEQUENCE ALIGNMENT OF THE HUMAN LAT ISOFORMS From analysing four isoforms of LAT our MSA shows large sections of the protein are conserved across all forms (shown by the amino acids highlighted in black). The amino acids that have been identified as key sites for signal transduction are preserved in all four isoforms indicating that they are able to carry out the same functions. Isoform A is the most common form of LAT protein found so for the next multiple sequence alignment we have just compared to this. MULTIPLE SEQUENCE ALIGNMENT OF THE LAT HOMOLOGUES IN DIFFERENT SPECIES From this MSA you can see there are many sections of the protein that are conserved across all four species, shown by the amino acids highlighted black. There are also individual amino acids highlighted grey showing that the sequence contains many amino acids with similar characteristics/properties to those in the same position on the different forms of LAT. This is important for binding to ensure the same regions always have distinct properties so can be recognised by their target substrates, for example residues with the binding site could be hydrophobic, negatively charged etc.. Using the predicted linear model we have produced, we have indicated the possible domains interacting with PLCγ1 , GRB2, GRAP2 and PIK3R1. These domains are conserved across all the alignments showing all the different forms of LAT bind to their target in the same regions. GRB2, GRAP2 and PIK3R1 all bind via their SH2 domain to the same region on the LAT protein [1]; this shows these proteins under certain conditions all recognise the same consensus sequence. SH2 domains recognise and bind to tyrosine phosphorylated sites. Looking at the highlighted interaction domains the tyrosine residue is always followed by Valine and Asparagine, hydrophobic and polar residues. We know the SH2 domain on GRB2 preferably binds to Y-X-N-X [2], where X is a hydrophobic residue (eg.Valine), which fits perfectly with all these alignments. PLC γ1 also interacts with phosphorylated tyrosine residues [1] by cleaving the phospholipid. From the interaction domain for PLC γ1 the tyrosine residue this time is followed by Valine and Leucine residues, which are both hydrophobic. The first four residues of LAT isoform a are the extracellular domain which are hydrophobic or negatively charged this is followed by the membrane spanning region (23 residues); these are mainly hydrophobic as well. The differences between any of the different forms of LAT could be due to alternative splicing; rearrangement of RNA exons produced during transcription of LAT gene. Both the LAT form found in mice and rats show huge sections that are missing compared to the Human isoform a and LAT found in cows, this could be due to a deletion due to the information coded not being required in the rat or mouse. A – indicates missing parts of the gene compared to other alignments. References [1]. Wange R.L, (2000). “LAT, the linker for activation of T cells: a bridge between T cell-specific and general signaling pathways”. Science's STKE : signal transduction knowledge environment, Vol.2000(63), p.re1. [2]. Nioche P, (2002). “Crystal structures of the SH2 domain of grb2: highlight on the binding of a new high-affinity inhibitor”. Journal of Molecular Biology, Vol.315(5), p.1167-1177.