Tyrosine phosphorylation of the Janus kinase 2 activation loop is essential for a high-activity catalytic state but dispensable for a basal catalytic state.
The phosphorylation of an "activation loop" within protein kinases is commonly associated with establishing catalytic competence, and phosphorylation of the Tyr(1007) residue in the activation loop of Janus kinase 2 (JAK2) has been shown to be essential for intracellular propagation of cytokine-initiated signaling. We provide evidence for the presence of a basal activity state of JAK2, which was observed in the absence of activation loop phosphorylation. Phosphorylation of the JAK2 activation loop was essential for conversion to the high-activity state, characterized by high-efficiency ATP utilization during autophosphorylation. Mutagenesis of activation loop tyrosine residues Tyr(1007/1008) to phenylalanine residues impaired, but did not abolish, the enzyme's ability to autophosphorylate. The activation loop mutant JAK2 could also transphosphorylate an inactive JAK2 fragment coexpressed in Sf21 cells, providing evidence of exogenous substrate phosphorylation. The mutant enzyme remained in a basal activity state characterized by low-efficiency ATP utilization during autophosphorylation. Mutagenesis of a critical Lys(882) residue to a glutamate residue abolished all evidence of kinase activity, confirming that the observed activity of Tyr-to-Phe mutants was not due to another kinase. Our data are consistent with the proposal that JAK2 is an inefficient but active enzyme in the absence of activation loop phosphorylation and is capable of conversion to a high-activity state by autophosphorylation under physiological ATP concentrations. This theoretically precludes the need for an upstream activating kinase. The activation process of JAK2 may be envisioned as a multistate process involving at least two kinetically distinct states of activity.
Chatti
Kiranam
K
Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, USA.
Farrar
William L
WL
Duhé
Roy J
RJ
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000502:physiology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D005822:Genetic Vectors; D053614:Janus Kinase 2; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000276:immunology / Q000378:metabolism; D011233:Precipitin Tests; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D011518:Proto-Oncogene Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics; D018411:Spodoptera / Q000235:genetics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C091973:autophosphorylation-dependent multifunctional protein kinase; D011505:Protein-Tyrosine Kinases; C507927:Jak2 protein, rat; D053614:Janus Kinase 2