Trk activation loop mutations

In search of mutations that mimic autophosphorylation

Toffalini and Demoulin (2010)  had some interesting comments on the need of phosphorylation of the 1-3 tyrosines on the Trk activation loop.  The most obvious effect of posphorylation would be the introduction of of a negative charge next to an arginine.  Mutations in the Trk activation loop have the potential to affect how the activation loop binds to and inhibits entrance of substrate to the active site in absence of phosphorylation.

cartoon showing Trk extracellular ligand binding sites on activation loop

Figure 1. The stereotypical receptor tyrosine kinase. Ligand binding induced dimerization results in phosphorylation of the activation loop and its movement from the active site.

Toffalini and Demoulin mentioned activation loop point mutations D816V and K642E in c-KIT.  Other activating mutations in the activation loop are presented in the supplemental data in this publication. In this review of  mutations in blood malignancies, c-KIT and FLT-3, were the RTK of interest.

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

Without prior knowledge of whether our cancer associated Trk mutations in the activation loop are drivers or  passengers, let us contemplate how an amino acid substitution might change the properties of the activation loop.

TrkA

The region of the activation loop as defined by the NCBI entry presented in Figure 2 was examined in COSMIC. Arrows point to autophosphorylated tyrosines. We will see the “DYYR” motif in the other Trk kinases. These amino acid substitutions could conceivably result in noticeable changes in the charge around the tyrosines in question. The pKa, or acid dissociation constant, is the pH at which half of the groups are in the ionized state. Arginines tend to be protonated at pH 7.5. The local pH of the entrance to the catalytic cleft might be totally different. Could some of these substitutions result in charge changes that would affect the interaction of the activation loop with the catalytic cleft in absence of phosphorylation?

 Cancer associated activation loop mutations in TrkA due to changes in amino acid charge

Figure 2 Cancer associated activation loop mutations in TrkA. Some of the mutations close to the autophosphorylation sites (arrows) could result in substantial changes in the charge.

 

Trk B

Note the “DYYR” motif that we saw before with TrkA. So far, not too many drastic cancer associated amino acid substitutions have appeared in the COSMIC database for the activation loop of TrkB.

 Cancer associated activation loop mutations in TrkB due to changes in amino acid charge

Figure 3 The activation loop of TrkB and COSMIC cancer associated single amino acid substitutions.

 

Trk C

Note yet another “DYYR” motif in Trk C. We have one record of a trytophan substitution, which of course cannot be phosphorylated. What is interesting about the activation loop of Trk C is that a threonine (T) for lysine (K) substitution has been observed seven nine in the cancer database.

 Cancer associated activation loop mutations in TrkC due to changes in amino acid charge

Figure 4. Some Trk C activation loop mutations associated with cancer.

 

Important Information

To the best of our knowledge, none of these Trk activation loop mutations result in kinase activity in the absence of autophosphorylation. Even if they did, they might not drive cancer when Trk is expressed at normal levels. There is a Trk inhibitor open clinical trial for cancers driven by Trk fusion proteins.