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. 1997 May;17(5):2497–2501. doi: 10.1128/mcb.17.5.2497

Activation of Jak2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop.

J Feng 1, B A Witthuhn 1, T Matsuda 1, F Kohlhuber 1, I M Kerr 1, J N Ihle 1
PMCID: PMC232098  PMID: 9111318

Abstract

The Janus protein tyrosine kinases (Jaks) play critical roles in transducing growth and differentiation signals emanating from ligand-activated cytokine receptor complexes. The activation of the Jaks is hypothesized to occur as a consequence of auto- or transphosphorylation on tyrosine residues associated with ligand-induced aggregation of the receptor chains and the associated Jaks. In many kinases, regulation of catalytic activity by phosphorylation occurs on residues within the activation loop of the kinase domain. Within the Jak2 kinase domain, there is a region that has considerable sequence homology to the regulatory region of the insulin receptor and contains two tyrosines, Y1007 and Y1008, that are potential regulatory sites. In the studies presented here, we demonstrate that among a variety of sites, Y1007 and Y1008 are sites of trans- or autophosphorylation in vivo and in in vitro kinase reactions. Mutation of Y1007, or both Y1007 and Y1008, to phenylalanine essentially eliminated kinase activity, whereas mutation of Y1008 to phenylalanine had no detectable effect on kinase activity. The mutants were also examined for the ability to reconstitute erythropoietin signaling in gamma2 cells, which lack Jak2. Consistent with the kinase activity, mutation of Y1007 to phenylalanine eliminated the ability to restore signaling. Moreover, phosphorylation of a kinase-inactive mutant (K882E) was not detected, indicating that Jak2 activation during receptor aggregation is dependent on Jak2 and not another receptor-associated kinase. The results demonstrate the critical role of phosphorylation of Y1007 in Jak2 regulation and function.

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Selected References

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  1. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  2. Brown M. T., Cooper J. A. Regulation, substrates and functions of src. Biochim Biophys Acta. 1996 Jun 7;1287(2-3):121–149. doi: 10.1016/0304-419x(96)00003-0. [DOI] [PubMed] [Google Scholar]
  3. Calalb M. B., Polte T. R., Hanks S. K. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol. 1995 Feb;15(2):954–963. doi: 10.1128/mcb.15.2.954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Darnell J. E., Jr, Kerr I. M., Stark G. R. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science. 1994 Jun 3;264(5164):1415–1421. doi: 10.1126/science.8197455. [DOI] [PubMed] [Google Scholar]
  5. Gauzzi M. C., Velazquez L., McKendry R., Mogensen K. E., Fellous M., Pellegrini S. Interferon-alpha-dependent activation of Tyk2 requires phosphorylation of positive regulatory tyrosines by another kinase. J Biol Chem. 1996 Aug 23;271(34):20494–20500. doi: 10.1074/jbc.271.34.20494. [DOI] [PubMed] [Google Scholar]
  6. Hubbard S. R., Wei L., Ellis L., Hendrickson W. A. Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature. 1994 Dec 22;372(6508):746–754. doi: 10.1038/372746a0. [DOI] [PubMed] [Google Scholar]
  7. Ihle J. N. Cytokine receptor signalling. Nature. 1995 Oct 19;377(6550):591–594. doi: 10.1038/377591a0. [DOI] [PubMed] [Google Scholar]
  8. Ihle J. N., Kerr I. M. Jaks and Stats in signaling by the cytokine receptor superfamily. Trends Genet. 1995 Feb;11(2):69–74. doi: 10.1016/s0168-9525(00)89000-9. [DOI] [PubMed] [Google Scholar]
  9. Ihle J. N., Witthuhn B. A., Quelle F. W., Yamamoto K., Silvennoinen O. Signaling through the hematopoietic cytokine receptors. Annu Rev Immunol. 1995;13:369–398. doi: 10.1146/annurev.iy.13.040195.002101. [DOI] [PubMed] [Google Scholar]
  10. Kohlhuber F., Rogers N. C., Watling D., Feng J., Guschin D., Briscoe J., Witthuhn B. A., Kotenko S. V., Pestka S., Stark G. R. A JAK1/JAK2 chimera can sustain alpha and gamma interferon responses. Mol Cell Biol. 1997 Feb;17(2):695–706. doi: 10.1128/mcb.17.2.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mohammadi M., Schlessinger J., Hubbard S. R. Structure of the FGF receptor tyrosine kinase domain reveals a novel autoinhibitory mechanism. Cell. 1996 Aug 23;86(4):577–587. doi: 10.1016/s0092-8674(00)80131-2. [DOI] [PubMed] [Google Scholar]
  12. Quelle F. W., Sato N., Witthuhn B. A., Inhorn R. C., Eder M., Miyajima A., Griffin J. D., Ihle J. N. JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region. Mol Cell Biol. 1994 Jul;14(7):4335–4341. doi: 10.1128/mcb.14.7.4335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Quelle F. W., Thierfelder W., Witthuhn B. A., Tang B., Cohen S., Ihle J. N. Phosphorylation and activation of the DNA binding activity of purified Stat1 by the Janus protein-tyrosine kinases and the epidermal growth factor receptor. J Biol Chem. 1995 Sep 1;270(35):20775–20780. doi: 10.1074/jbc.270.35.20775. [DOI] [PubMed] [Google Scholar]
  14. Superti-Furga G., Courtneidge S. A. Structure-function relationships in Src family and related protein tyrosine kinases. Bioessays. 1995 Apr;17(4):321–330. doi: 10.1002/bies.950170408. [DOI] [PubMed] [Google Scholar]
  15. Taniguchi T. Cytokine signaling through nonreceptor protein tyrosine kinases. Science. 1995 Apr 14;268(5208):251–255. doi: 10.1126/science.7716517. [DOI] [PubMed] [Google Scholar]
  16. Taylor S. J., Shalloway D. Src and the control of cell division. Bioessays. 1996 Jan;18(1):9–11. doi: 10.1002/bies.950180105. [DOI] [PubMed] [Google Scholar]
  17. Ullrich A., Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. doi: 10.1016/0092-8674(90)90801-k. [DOI] [PubMed] [Google Scholar]
  18. Wang D., Stravopodis D., Teglund S., Kitazawa J., Ihle J. N. Naturally occurring dominant negative variants of Stat5. Mol Cell Biol. 1996 Nov;16(11):6141–6148. doi: 10.1128/mcb.16.11.6141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Watling D., Guschin D., Müller M., Silvennoinen O., Witthuhn B. A., Quelle F. W., Rogers N. C., Schindler C., Stark G. R., Ihle J. N. Complementation by the protein tyrosine kinase JAK2 of a mutant cell line defective in the interferon-gamma signal transduction pathway. Nature. 1993 Nov 11;366(6451):166–170. doi: 10.1038/366166a0. [DOI] [PubMed] [Google Scholar]
  20. Wilks A. F., Harpur A. G. Cytokine signal transduction and the JAK family of protein tyrosine kinases. Bioessays. 1994 May;16(5):313–320. doi: 10.1002/bies.950160505. [DOI] [PubMed] [Google Scholar]
  21. Witthuhn B. A., Quelle F. W., Silvennoinen O., Yi T., Tang B., Miura O., Ihle J. N. JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell. 1993 Jul 30;74(2):227–236. doi: 10.1016/0092-8674(93)90414-l. [DOI] [PubMed] [Google Scholar]
  22. Yamamoto K., Quelle F. W., Thierfelder W. E., Kreider B. L., Gilbert D. J., Jenkins N. A., Copeland N. G., Silvennoinen O., Ihle J. N. Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation. Mol Cell Biol. 1994 Jul;14(7):4342–4349. doi: 10.1128/mcb.14.7.4342. [DOI] [PMC free article] [PubMed] [Google Scholar]

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