Other Kinases Can Substitute for Jak2 in Signal Transduction by Interferon-γ

Serguei V Kotenko,Lara S Izotova,Brian P Pollack,Geetha Muthukumaran,Kirsi Paukku,Olli Silvennoinen,James N Ihle,Sidney Pestka

doi:10.1074/jbc.271.29.17174

Abstract

Each cytokine which utilizes the Jak-Stat signal transduction pathway activates a distinct combination of members of the Jak and Stat families. Thus, either the Jaks, the Stats, or both could contribute to the specificity of ligand action. With the use of chimeric receptors involving the interferon gamma receptor (IFN-gammaR) complex as a model system, we demonstrate that Jak2 activation is not an absolute requirement for IFN-gamma signaling. Other members of the Jak family can functionally substitute for Jak2. IFN-gamma can signal through the activation of Jak family members other than Jak2 as measured by Statlalpha homodimerization and major histocompatibility complex class I antigen expression. This indicates that Jaks are interchangeable and indiscriminative in the Jak-Stat signal transduction pathway. The necessity for the activation of one particular kinase during signaling can be overcome by recruiting another kinase to the receptor complex. The results may suggest that the Jaks do not contribute to the specificity of signal transduction in the Jak-Stat pathway to the same degree as Stats.

Highlights

The receptor chains oligomerize allowing the associated kinases to interact and likely cross-activate each other by tyrosine phosphorylation
The intracellular domain of interferon ␥ (IFN-␥)R2 associates with Jak2 and brings Jak2 into the complex upon ligand binding (Kotenko et al, 1995; Sakatsume et al, 1995)
The expression of a kinase negative Jak2 mutant in a Jak2 negative (␥2A) cell line cannot sustain an IFN-␥ response (Briscoe et al, 1995), indicating that the protein tyrosine kinases (PTK) activity of Jak2 is absolutely necessary for IFN-␥ signaling

Summary

EXPERIMENTAL PROCEDURES

Restriction Endonucleases, and Other Enzymes—Taq polymerase and all restriction endonucleases were from Boehringer Mannheim Biochemicals or New England Biolabs; Sequenase 2.0 and T4 DNA ligase were from U. The PCR product was digested with EcoRI restriction endonuclease and ligated into EcoRI and blunt ended BamHI sites of the pcDNA3 vector. The CRFB4 cDNA was released from one of the rescued plasmids designated pCEV15CRFB4 –1 by digestion with ThaI and SalI restriction endonucleases and cloned into EcoRV and XhoI sites of the pcDNAIneo vector (Invitrogen). The final ␥R2/␥R1 PCR product was digested with BstEII restriction endonuclease and cloned into BstEII and blunt-ended XbaI sites of the p␥R2 plasmid. To introduce an NheI site in the beginning of the transmembrane domain of the Hu-IFN-␥R2 cDNA clone, the PCR reaction was performed with two primers 5Ј-GCCTTTTTTAGTTATTATGTC-3Ј and 5ЈATCGCTAGCCATTGCTGAAGCTCAGTGGAGG-3Ј and plasmid p␥R2 as a template according to a standard protocol (Sambrook et al, 1989). The IL-2R␥C PCR product was digested with NheI restriction endonuclease and ligated into the NheI and blunt ended XbaI sites of plasmid p␥R2NheI. COS-1 cells were transiently transfected with the expression vectors by the DEAE-dextran procedure with dimethyl sulfoxide shock (Seed and Aruffo, 1987; Sussman and Milman, 1984)

Signal Transduction of Cytokine Receptors

RESULTS

DISCUSSION