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

Abstract

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 upregulation (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 Tyr 248 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.

Proteomics analysis of mitochondrial proteins reveals overexpression of a mitochondrial protein chaperon, prohibitin, in cells expressing hepatitis C virus core protein†

“…If the degradation is mediated by ubiquitin as reported, 23 it is possible that the interaction with the core protein interferes with ubiquitin-binding and protects prohibitin from degradation by proteasome. Some posttranslational protein modifications such as phosphorylation are other possible factors for the stabilization, because prohibitin can be serine-phosphorylated 32 ; however, in our examination no serine/threonine/tyrosine phosphorylation of prohibitin was detected in core-expressing cells (data not shown). Thus far, there are no studies showing that prohibitin stabilization leads to a suppressed function as a mitochondrial chaperon.…”

Prohibitin has an important role in adipocyte differentiation

“…48 In this context it is important to note that PHB has been reported to provide protection against oxidative damage, and we have reported that PHB functions as an iron-binding protein and is involved in mitochondrial iron homeostasis. 47,49 Furthermore, the downregulation of PHB protein level in adipose tissues from ob/ob mice and upregulation of PHB in response to leptin in 3T3-L1 adipocytes further support a role of PHB in adipose tissue homeostasis. Upregulation of PHB by insulin and an inhibitory effect of PHB overexpression on insulin-induced adipogenesis may represent a negative feedback relationship between insulin and PHB to maintain each other's function, and subsequently adipose tissue homeostasis under normal condition.…”

Prohibitin-2 Binding Modulates Insulin-like Growth Factor-binding Protein-6 (IGFBP-6)-induced Rhabdomyosarcoma Cell Migration

“…The PHB1⅐PHB2 phosphocomplex is required for mitochondrial homeostasis and survival of human T cells (47). Insulin-induced phosphorylation of cell membrane PHB1 at tyrosine 114 and 259 by insulin receptor kinase facilitates its interaction with Shp1, which in turn negatively regulates PIP3/Akt signaling in myoblasts (48) and breast cancer cells (48,49).…”