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Molecular and Cellular Biology, July 2009, p. 3710-3721, Vol. 29, No. 13
0270-7306/09/$08.00+0     doi:10.1128/MCB.01869-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Targeted Knock-In Mice Expressing Mutations of CD28 Reveal an Essential Pathway for Costimulation{triangledown} ,{dagger}

Lindzy F. Dodson,1,2,{ddagger} Jonathan S. Boomer,2,{ddagger} Christine M. Deppong,2 Dulari D. Shah,2 Julia Sim,3 Traci L. Bricker,2 John H. Russell,3 and Jonathan M. Green1,2*

Division of Biology and Biomedical Sciences,1 Department of Internal Medicine,2 Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri 631103

Received 8 December 2008/ Returned for modification 30 January 2009/ Accepted 14 April 2009

Despite extensive study, the role of phosphatidylinositol 3-kinase (PI3-kinase) activation in CD28 function has been highly contentious. To definitively address this question, we generated knock-in mice expressing mutations in two critical domains of the cytoplasmic tail of CD28. Mutation of the proximal tyrosine motif interrupted PI3-kinase binding and prevented CD28-dependent phosphorylation of protein kinase B (PKB)/Akt; however, there was no detectable effect on interleukin-2 (IL-2) secretion, expression of Bcl-XL, or on T-cell function in vivo. Furthermore, we demonstrate that signaling initiated by the C-terminal proline motif is directly responsible for tyrosine phosphorylation of phosphoinosotide-dependent kinase 1, protein kinase C{theta}, and glycogen synthase kinase 3β, as well as contributing to threonine phosphorylation of PKB. T cells mutated in this domain were profoundly impaired in IL-2 secretion, and the mice had marked impairment of humoral responses as well as less severe disease manifestations in experimental allergic encephalomyelitis. These data demonstrate that the distal proline motif initiates a critical nonredundant signaling pathway, whereas direct activation of PI3-kinase by the proximal tyrosine motif of CD28 is not required for normal T-cell function.

* Corresponding author. Mailing address: 660 S. Euclid Ave., P.O. Box 8052, St. Louis, MO 63110. Phone: (314) 747-3591. Fax: (314) 362-8987. E-mail: jgreen{at}wustl.edu

{triangledown} Published ahead of print on 27 April 2009.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

{ddagger} L.F.D. and J.S.B. contributed equally to this work and should be considered co-first authors.

Molecular and Cellular Biology, July 2009, p. 3710-3721, Vol. 29, No. 13
0270-7306/09/$08.00+0     doi:10.1128/MCB.01869-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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