title: Crystal structure of the ALK (anaplastic lymphoma kinase) catalytic domain.
abstract: 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.
authors.affiliation: The Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA. clee@gnf.org
authors.forename: Christian C
authors.initials: CC
authors.lastname: Lee
authors.forename: Yong
authors.initials: Y
authors.lastname: Jia
authors.forename: Nanxin
authors.initials: N
authors.lastname: Li
authors.forename: Xiuying
authors.initials: X
authors.lastname: Sun
authors.forename: Kenneth
authors.initials: K
authors.lastname: Ng
authors.forename: Eileen
authors.initials: E
authors.lastname: Ambing
authors.forename: Mu-Yun
authors.initials: MY
authors.lastname: Gao
authors.forename: Su
authors.initials: S
authors.lastname: Hua
authors.forename: Connie
authors.initials: C
authors.lastname: Chen
authors.forename: Sungjoon
authors.initials: S
authors.lastname: Kim
authors.forename: Pierre-Yves
authors.initials: PY
authors.lastname: Michellys
authors.forename: Scott A
authors.initials: SA
authors.lastname: Lesley
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Harris
authors.forename: Glen
authors.initials: G
authors.lastname: Spraggon
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000077548:Anaplastic Lymphoma Kinase; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism; D009447:Neuroblastoma / Q000201:enzymology / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D019311:Staurosporine / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D050505:Mutant Proteins; D000244:Adenosine Diphosphate; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D019311:Staurosporine