Autophosphorylation of JAK2 on Tyrosines 221 and 570 Regulates Its Activity

Abstract

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2.

We thank Romano T. Kroemer for providing the coordinates for the JAK2 model (16, 30) generated in his laboratory.

This work was supported by NIH grant DK34171 (to C.C.-S.) and a grant from the Danish Basic Research Foundation (to O.N.J.). Oligonucleotides were synthesized by the Biomedical Research Core Facility at the University of Michigan with support from the Michigan Diabetes Research and Training Center (P60-DK20572), the University of Michigan Multipurpose Arthritis Center (P60-AR20557), and the University of Michigan Comprehensive Cancer Center (NIH P30 CA46592). cDNA sequencing was supported by the Cellular and Molecular Biology Core of the Michigan Diabetes Research and Training Center.