Trk kinase domain nonsense mutations

A non-concern:  nonsense mutations in the TRK kinase domain?

As we have reviewed earlier, nonsense mutations create premature translation stop condons. The end result is a truncated Trk that may or may not involve the active site. Toffalini and Demoulin (2010) have reviewed mutations in receptor tyrosine kinases (RTK) involved in blood malignancies. Trk kinases were not covered in this review, but many of the same concepts may apply.

cartoon showing Trk extracellular ligand binding active sites on activation loop

Figure 1. The kinase domain not only contains the active site and the activation loop, but also docking sites for signaling molecules

Toffalini F, Demoulin JB.(2010) New insights into the mechanisms of hematopoietic cell transformation by activated receptor tyrosine kinases. Blood. 116(14):2429-37. PubMed

The activation loop of the stereotypical receptor tyrosine  kinase (RTK) blocks entry of substrate and ATP from the active site between the two lobes. In RTK, binding of the extracellular domain to a ligand destabilizes the inhibitory interaction of the juxta membrane region with the C-terminal lobe of the kinase domain (Tofalini and Demoulin 2010). Phosphorylation of the activation loop removes it from the cleft between the two lobes of the kinase domain. Other sites on the kinase may target the stereotypical three RTK signaling pathway substrates to the active site. Let’s think about this as we review some of the cancer associated mutations that result in premature translation termination of Trk kinases.

Other references for this blog include NCBI, RCSB.org with links to UniProt.org, and COSMIC.

Trk A

Only the E755* nonsense mutation (panel c) spares the ATP and peptide binding sites as well as the activation loop (panel a). The W711* nonsense mutation (panel c) takes out a small portion of the peptide binding site (panel a). Both the W711* and E755* nonsense mutations take out at least phosphorylation sites. Y791 is not only a tyrosine phosphorylation site but also a PLCγ1 docking site UniProt. Consult UniProt for the numerous TrkA mutations associated with CIPA.

TrkA kinase domain nonsense mutations diagram showing ATP binding site, polypeptide binding site and activation loop

Figure 2 TrkA kinase domain nonsense mutations a. domain information and sequence from NCBI b. Phosphorylation sites from RCSB/UniProt c. Nonsense mutations from COSMIC

 

The other nonsense mutations probably result in an inactive enzyme.

 

Trk B

COSMIC lists three kinase domain nonsense mutations that miss the important ATP and peptide binding motifs as well as the activation loop. These are %790*, W806*, and R808* (panel c) spares the ATP and peptide binding sites as well as the activation loop (panel a). The W711* nonsense mutation (panel c). Y817 is not only a tyrosine phosphorylation site but also a PLCγ1 docking site UniProt that would be removed by all three of these nonsense mutations.

TrkB kinase domain nonsense mutations diagram showing ATP binding site, polypeptide binding site and activation loop

Figure 3 TrkB kinase domain nonsense mutations a. domain information and sequence from NCBI b. Phosphorylation sites from RCSB/UniProt c. Nonsense mutations from COSMIC

 

We do not know the impact of these nonsense mutations on cancer progression and cell signaling.

Trk C

Two regions seem to compose the activation loop of TrkC: G696-R711 and V726-R735 (panel a). W757*, Q759*, and C768* leave the ATP binding site and activation loops and most of the peptide binding site (panel a and c). UniProt (UP) lists a phosphorylation site at Y834 that is also a PLCγ1 docking site (panel b), just like TrkA and TrkB.

TrkC kinase domain nonsense mutations diagram showing ATP binding site, polypeptide binding site and activation loop

TrkC kinase domain nonsense mutations a. domain information and sequence from NCBI b. Phosphorylation sites from RCSB/UniProt c. Nonsense mutations from COSMIC

 

Interesting Trk mutations may be a distraction from what matters

What would happen to Trk kinase activity if far C-terminal portions of the protein, separate from the active site, were removed? An interesting philosophical question that is removed from what are issues that warrant action in treating cancer.

  1. Are these mutant proteins even expressed in the cancer?
  2. If truncated kinases are still regulated by ligand binding, are the ligands like NGF even present in the tumor?
  3. If PLCγ docking were obliterated while leaving normal docking of other substrates, would this impact cancer?

 

What really matters is how much active kinase is being produced and whether this kinase is functioning unregulated. This is the case for TRK fusions. There is a simple test to detect Trk fusions that drive cancers. There is also an open clinical trial testing a specific Trk inhibitor.