ALK (
anaplastic lymphoma kinase) is an RTK (
receptor tyrosine kinase) of the IRK (
insulin receptor kinase) superfamily, which share an YXXXYY autophosphorylation motif within their A-loops (activation loops). A common activation and regulatory mechanism is believed to exist for members of this superfamily typified by IRK and IGF1RK (
insulin-like growth factor receptor kinase-1).
Chromosomal translocations involving ALK were first identified in
anaplastic large-cell lymphoma, a subtype of
non-Hodgkin's lymphoma, where aberrant fusion of the
ALK kinase domain with the NPM (
nucleophosmin) dimerization domain results in autophosphosphorylation and
ligand-independent activation. Activating mutations within the full-length
ALK kinase domain, most commonly R1275Q and F1174L, which play a major role in
neuroblastoma, were recently identified. To provide a structural framework for understanding these mutations and to guide structure-assisted drug discovery efforts, the X-ray crystal structure of the unphosphorylated ALK catalytic domain was determined in the apo,
ADP- and
staurosporine-bound forms. The structures reveal a partially inactive
protein kinase conformation distinct from, and lacking, many of the negative regulatory features observed in inactive IGF1RK/IRK structures in their unphosphorylated forms. The A-loop adopts an inhibitory pose where a short proximal A-loop helix (alphaAL) packs against the alphaC helix and a novel N-terminal beta-turn motif, whereas the distal portion obstructs part of the predicted
peptide-binding region. The structure helps explain the reported unique
peptide substrate specificity and the importance of phosphorylation of the first A-loop Tyr1278 for
kinase activity and
NPM-ALK transforming potential. A single
amino acid difference in the ALK substrate
peptide binding P-1 site (where the P-site is the phosphoacceptor site) was identified that, in conjunction with A-loop sequence variation including the RAS (Arg-Ala-Ser)-motif, rationalizes the difference in the A-loop
tyrosine autophosphorylation preference between ALK and IGF1RK/IRK. Enzymatic analysis of recombinant R1275Q and F1174L ALK mutant catalytic domains confirms the enhanced activity and transforming potential of these mutants. The transforming ability of the full-length ALK mutants in soft
agar colony growth assays corroborates these findings. The availability of a three-dimensional structure for ALK will facilitate future structure-function and rational drug design efforts targeting this
receptor tyrosine kinase.