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J. Biol. Chem., Vol. 283, Issue 8, 4699-4713, February 22, 2008

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The PHB1/2 Phosphocomplex Is Required for Mitochondrial Homeostasis and Survival of Human T Cells^*

From the Department of Biological Sciences, University of Texas, El Paso, Texas 79902

Many immune pathologies are the result of aberrant regulation of T lymphocytes. A functional proteomics approach utilizing two-dimensional gel electrophoresis coupled with mass spectrometry was employed to identify differentially expressed proteins in response to T cell activation. Two members of the prohibitin family of proteins, Phb1 and Phb2, were determined to be up-regulated 4–5-fold upon activation of primary human T cells. Furthermore, their expression was dependent upon CD3 and CD28 signaling pathways that synergistically led to the up-regulation (13–15-fold) of Phb1 and Phb2 mRNA levels as early as 48 h after activation. Additionally, orthophosphate labeling coupled with phosphoamino acid analysis identified Phb1 to be serine and Phb2 serine and tyrosine phosphorylated. Tyrosine phosphorylation of Phb2 was mapped to Tyr²⁴⁸ using mass spectrometry and confirmed by mutagenesis and phosphospecific antibodies. In contrast to previous reports of Phb1 and Phb2 being nuclear localized, subcellular fractionation, immunofluorescent, and electron microscopy revealed both proteins to localize to the mitochondrial inner membrane of human T cells. Accordingly, small interfering RNA-mediated knockdown of Phbs in Kit225 cells resulted in disruption of mitochondrial membrane potential. Additionally, Phb1 and Phb2 protein levels were up-regulated 2.5-fold during cytokine deprivation-mediated apoptosis of Kit225 cells, suggesting this complex plays a protective role in human T cells. Taken together, Phb1 and Phb2 are novel phosphoproteins up-regulated during T cell activation that function to maintain mitochondrial integrity and thus represent previously unrecognized therapeutic targets for regulating T cell activation, differentiation, viability, and function.

Received for publication, October 3, 2007 , and in revised form, November 14, 2007.

^* This work was supported by National Institutes of Health Grant AI053566 (to R. A. K.) and 5G12RR008124 from the National Center for Research Resources, a component of the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biological Sciences, 500 W. University Ave., El Paso, TX 79902. Tel.: 915-747-5844; Fax: 915-747-5808; E-mail: rkirken{at}utep.edu.

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