Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1997 Jun 24;94(13):6910-5.
doi: 10.1073/pnas.94.13.6910.

The amino terminus of JAK3 is necessary and sufficient for binding to the common gamma chain and confers the ability to transmit interleukin 2-mediated signals

M Chen  1 , A Cheng, Y Q Chen, A Hymel, E P Hanson, L Kimmel, Y Minami, T Taniguchi, P S Changelian, J J O'Shea
Affiliations

The amino terminus of JAK3 is necessary and sufficient for binding to the common gamma chain and confers the ability to transmit interleukin 2-mediated signals

M Chen et al. Proc Natl Acad Sci U S A. .

Abstract

JAK3 is a protein tyrosine kinase that specifically associates with the common gamma chain (gammac), a shared subunit of receptors for interleukin (IL) 2, 4, 7, 9, and 15. Patients deficient in either JAK3 or gammac presented with virtually identical forms of severe combined immunodeficiency (SCID), underscoring the importance of the JAK3-gammac interaction. Despite the key roles of JAK3 and gammac in lymphocytic development and function, the molecular basis of this interaction remains poorly understood. In this study, we have characterized the regions of JAK3 involved in gammac association. By developing a number of chimeric JAK3-JAK2 constructs, we show that the binding specificity to gammac can be conferred to JAK2 by transferring the N-terminal domains of JAK3. Moreover, those JAK3-JAK2 chimeras capable of binding gammac were also capable of reconstituting IL-2 signaling as measured by inducible phosphorylation of the chimeric JAK3-JAK2 protein, JAK1, the IL-2 receptor beta chain, and signal transducer and activator of transcription 5A. Subsequent deletion analyses of JAK3 have identified the N-terminal JH7-6 domains as a minimal region sufficient for gammac association. Furthermore, expression of the mutant containing only the JH7-6 domains effectively competed with full-length JAK3 for binding to gammac. We conclude that the JH7-6 domains of JAK3 are necessary and sufficient for gammac association. These studies offer clues toward a broader understanding of JAK-mediated cytokine signaling and may provide a target for the development of novel therapeutic modalities in immunologically mediated diseases.

PubMed Disclaimer

Figures

Figure 2
Figure 2
The binding specificity of JAK3 to γc can be conferred to JAK2 by transferring the N-terminal domains. (A) A schematic representation of the J3J2 chimeras. J3(7-3)J2(2-1) contains the N-terminal JH7-3 domains from JAK3 (aa 1–519) fused to the C-terminal JH2-1 domains of JAK2 (aa 543–1130). J3(7-4)J2(4-1) contains the N-terminal JH7 through a portion of JH4 domains of JAK3 (aa 1–370) fused to the C-terminal JH4-1 domains of JAK2 (aa 395–1130). J3(7)J2(6-1) contains the N-terminal JH7 domain of JAK3 (aa 1–124) fused to the C-terminal JH6-1 domains of JAK2 (aa 141–1130). J3(N)J2(7-1) contains the extreme N terminus (aa 1–40) of JAK3 fused to the JH7-1 domains of JAK2 (aa 55–1130). (B) Association of J3J2 chimeras with the cytoplasmic domain of γc. COS cells were transfected with 3 μg of the indicated cDNAs. These cell lysates were immunoprecipitated with anti-Tac and blotted with anti-γc (Middle) and anti-JAK2 that crossreacts with JAK3 (Top). Cell lysates were analyzed for expression of the transfected JAK constructs by immunoblotting with anti-JAK2 (Bottom).
Figure 4
Figure 4
The JAK3 N terminus is necessary and sufficient for γc binding. (A) A schematic representation of the JAK3 C-terminal and N-terminal deletion mutants. The numbers indicate the last (C-terminal) or the second (N-terminal) aa residue in each mutant with respect to the original JAK3 sequence. (B-D) COS cells were transfected with 5 μg of the indicated cDNAs. Lysates were immunoprecipitated with anti-Tac and blotted with anti-JAK3(N) (B and C Top) or anti-JAK3 C-terminal [anti-JAK3(C)] (D Top) and anti-γc (Middle). Expression levels of various mutants were analyzed by immunoblotting with anti-JAK3(N) (B and C Bottom) or anti-JAK3(C) (D Bottom).
Figure 1
Figure 1
JAK3 specifically associates with the cytoplasmic domain of γc. COS cells were transfected by the DEAE–dextran method with 5 μg of the indicated cDNAs. Cell lysates were immunoprecipitated (IP) with anti-Tac mAb and immunoblotted (IB) with antisera to JAK3 (lanes 1–4), JAK1 (lanes 5–8), or JAK2 (lanes 9–12). The levels of expression of JAK1, JAK2, or JAK3 were assessed by immunoblotting with corresponding antibodies (Bottom).
Figure 3
Figure 3
Reconstitution of IL-2-dependent signaling in IL-2R-expressing fibroblast cells requires physical coupling of J3J2 chimeras to γc. Serum-starved cells (1.5 × 107 cells) were left unstimulated or stimulated with IL-2 (1,000 units/ml) as indicated for 15 min; cell lysates were immunoprecipitated with anti-JAK2 (A), anti-STAT5A (B), or anti-IL-2Rβ (561) (C). Tyrosine phosphorylation was assayed by blotting with anti-phosphotyrosine (anti-PTyr) (Top). Membranes were reprobed with the anti-JAK3 N-terminal [anti-JAK3(N)] (A), anti-STAT5A (B), or anti-IL-2Rβ (ErdA) (C), to confirm equal loading (Bottom).
Figure 5
Figure 5
The JAK3 N terminus competes with full-length JAK3 for binding to γc. COS cells were cotransfected with JAK3 (0.5 μg) and Tacγc (0.5 μg) together with the vector pME18s (5 μg) (lane 1), JAK2 (5 μg) (lane 2), or 1, 2, or 5 μg of J3(StuI) (lanes 3–5, respectively) or J3(ΔJH1) (lanes 6–8, respectively). These cell lysates were immunoprecipitated with anti-Tac and then blotted with anti-JAK3(N) (Left Upper) or anti-γc (Left Lower). The expression levels of various JAK3 mutants and JAK2 were analyzed by immunoblotting with anti-JAK3(N) (Right Upper) and anti-JAK2 (Right Lower), which immunoblots both JAK2 and JAK3. The unspecified middle band in all lanes shown on the anti-JAK2 blot is consistently recognized by this antibody as described by the manufacturer.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Paul W E, Seder R A. Cell. 1994;76:241–251. - PubMed
    1. Ihle J N, Witthuhn B A, Quelle F W, Yamamoto K, Silvennoinen O. Annu Rev Immunol. 1995;13:369–398. - PubMed
    1. Taniguchi T. Science. 1995;268:251–255. - PubMed
    1. Schindler C, Darnell J E J. Annu Rev Biochem. 1995;64:621–651. - PubMed
    1. Darnell J E J, Kerr I M, Stark G R. Science. 1994;264:1415–1421. - PubMed

MeSH terms

LinkOut - more resources