Suresh Mishra

The Prohibitins: emerging roles in diverse functions

The prohibitins, Phb1 and Phb2 are highly conserved proteins in eukaryotic cells that are present in multiple cellular compartments. Initial investigations focused on the role of Phb1 as an inhibitor of cell proliferation hence the original name prohibitin. However both proteins appear to have a diverse range of functions and recent evidence suggests that the prohibitins have very similar but as yet only partially understood functions. In addition to their role as chaperone proteins in the mitochondria, and their ability to target to lipid rafts, their is now compelling evidence that both prohibitins are localized in the nucleus and can modulate transcriptional activity by interacting with various transcription factors, including the steroid hormone receptors, either directly or indirectly. In addition Phb1 and Phb2 are present in the circulation and can be internalized when added to cultured cells suggesting that the circulating prohibitins may have some regulatory role. This review presents some of the recent developments in prohibitin research and focuses on the similarities in the structure and function of these interesting proteins.

The role of prohibitin in cell signaling.

Prohibitin‐1 (PHB, also known as PHB1), a member of the Band‐7 family of proteins, is highly conserved evolutionarily, widely expressed, and present in different cellular compartments. Genetic studies with different organism models have provided strong evidence for an important biological role of PHB in mitochondrial function, cell proliferation, and development. Recent discoveries regarding the involvement of PHB in phophatidylinositol‐3‐kinase (PI3K)/protein kinase B (Akt) and transforming growth factor‐β (TGF‐β)/signal transducers and activators of transcription signaling pathways, and earlier reports on the interaction of PHB with Raf and its critical role in Ras/mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) signaling opened up the possibility that PHB has functions outside of the mitochondria (extramitochondrial) and may be a multifunctional protein. The PI3K/Akt and Ras/MAPK/ERK signaling cascades are versatile signaling processes that diverge from the same receptor tyrosine kinase root, and are involved in cell metabolism, proliferation, and development. Here, we review the emerging role of PHB and its post‐translational modifications in signal transduction pathways, especially in PI3K/Akt and Ras/MAPK/ERK signaling. A recent discovery of opposing effects of PHB on longevity under different metabolic states and its potential connection with insulin/insulin‐like growth factor‐I signaling is also discussed.

Perturbations in bone formation and resorption in insulin-like growth factor binding protein-3 transgenic mice.

IGF‐I and their binding proteins are important in bone health. Examination of BMD, osteoblast proliferation, and markers of bone resorption in transgenic mice that constitutively overexpress IGFBP‐3 indicates that overexpression of IGFBP‐3 increases osteoclast number and bone resorption, impairs osteoblast proliferation, and has a significant negative effect on bone formation.

Phosphorylation of histones by tissue transglutaminase

Tissue transglutaminase 2 (TG2) has recently been shown to have intrinsic serine/threonine kinase activity. Since histones are known to be cross-linked by TG2, we investigated whether histones are also substrates for TG2 kinase activity. TG2 was able to phosphorylate H1, H2A, H2B, H3, and H4 histones in vitro. Using peptide substrates and phosphospecific antibodies we demonstrated that TG2 phosphorylated Ser10 in H3 and that this phosphorylation was reduced by acetylation, whereas phosphorylation of Ser10 by TG2 enhanced acetylation. Furthermore we demonstrated that exogenous TG2 phosphorylated H1 and H3 in nucleosome preparations. We examined the abundance of TG2 in DNA-associated proteins from MCF-7 cells treated with phorbol ester (TPA) and 17β-estradiol (E2). TG2 abundance was significantly reduced in E2-treated cells and enhanced in TPA-treated cells. In summary we have demonstrated that TG2 is able to phosphorylate purified histone proteins, and H3 and H1 in chromatin preparations, and it is associated with chromatin in breast cancer cells. These studies suggest a novel role for TG2 in the regulation of chromatin structure and function.

Transglutaminase 2 kinase activity facilitates protein kinase A-induced phosphorylation of retinoblastoma protein.

Transglutaminase 2 (TG2, tissue transglutaminase) is a multifunctional protein involved in cross-linking a variety of proteins, including retinoblastoma protein (Rb). Here we show that Rb is also a substrate for the recently identified serine/threonine kinase activity of TG2 and that TG2 phosphorylates Rb at the critically important Ser780 residue. Furthermore, phosphorylation of Rb by TG2 destabilizes the Rb·E2F1 complex. TG2 phosphorylation of Rb was abrogated by high Ca2+ concentrations, whereas TG2 transamidating activity was inhibited by ATP. TG2 was itself phosphorylated by protein kinase A (PKA). Phosphorylation of TG2 by PKA attenuated its transamidating activity and enhanced its kinase activity. Activation of PKA in mouse embryonic fibroblasts (MEF) with dibutyryl-cAMP enhanced phosphorylation of both TG2 and Rb by a process that was inhibited by the PKA inhibitor H89. Treatment with dibutyryl-cAMP enhanced Rb phosphorylation in MEFtg2+/+ cells but not in MEFtg2–/– cells. These data indicate that Rb is a substrate for TG2 kinase activity and suggest that phosphorylation of Rb, which results from activation of PKA in fibroblasts, is indirect and requires TG2 kinase activity.

Prohibitin attenuates insulin‐stimulated glucose and fatty acid oxidation in adipose tissue by inhibition of pyruvate carboxylase

Prohibitin (PHB‐1) is a highly conserved protein involved in mitochondrial biogenesis and function. It is secreted in lipid droplets from adipocytes and is present in the circulation. In adipose tissue it functions as a membrane receptor and can target binding partners to the mitochondria. Here we report that PHB‐1 has a hitherto undescribed role as an inhibitor of pyruvate carboxylase (PC). As a consequence, it can modulate insulin‐stimulated glucose and fatty acid oxidation. It had no effect on insulin‐stimulated 2‐deoxglucose uptake by isolated adipocytes but inhibited insulin‐stimulated oxidation of [14C]glucose with a half‐maximal concentration of ≈ 4 nm. It also inhibited oleic acid oxidation in glucose‐depleted adipocytes via depletion of oxaloacetate. In vitro experiments using broken‐cell assays confirmed that PHB‐1 inhibited PC. MALDI‐TOF analysis of proteins identified by cross‐linking of PHB‐1 to adipocyte membranes indicated that PHB‐1 is closely associated with PC and EH domain 2 (EHD2). On the basis of these data, we propose that PHB‐1 is recycled between the extracellular space and the mitochondria by a mechanism involving lipid rafts and EHD2 and can modulate mitochondrial fuel metabolism by inhibition of PC.

O-GlcNAc modification: why so intimately associated with phosphorylation?

Post-translational modification of proteins at serine and threonine side chains by β-N-acetylglucosamine (O-GlcNAc) mediated by the enzyme β-N-acetylglucosamine transferase has been emerging as a fundamental regulatory mechanism encompassing a wide range of proteins involved in cell division, metabolism, transcription and cell signaling. Furthermore, an extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in a variety of proteins has been reported to exist. However, our understanding of the regulatory mechanisms involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins is still elusive. Recent success in the mapping of O-GlcNAc modification sites in proteins as a result of technological advancement in mass spectrometry have revealed two important clues which may be inherently connected to the regulation of O-GlcNAc modification and its interplay with phosphorylation in proteins. First, almost all O-GlcNAc modified proteins are known phospho proteins. Second, the prevalence of tyrosine phosphorylation among O-GlcNAc modified proteins is exceptionally higher (~68%) than its normal occurrence (~2%) alone. We hypothesize that phosphorylation may be a requisite for O-GlcNAc modification and tyrosine phosphorylation plays a role in the interplay between O-GlcNAc modification and serine/threonine phosphorylation in proteins. In other words, the interplay between O-GlcNAc modification and phosphorylation is not limited to serine/threonine phosphorylation but also includes tyrosine phosphorylation. Our hypothesis provides an opportunity to understand the underlying mechanism involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins. Furthermore, implication of our hypothesis extends to tyrosine kinase signaling.

Prohibitin is expressed in pancreatic β‐cells and protects against oxidative and proapoptotic effects of ethanol

Pancreatic β‐cell dysfunction is a prerequisite for the development of type 2 diabetes. Alcoholism is a diabetes risk factor and ethanol increases oxidative stress in β‐cells, whereas the mitochondrial chaperone prohibitin (PHB) has antioxidant effects in several cell types. In the present study we investigated whether PHB is expressed in β‐cells and protects these cells against deleterious effects of ethanol, using INS‐1E and RINm5F β‐cell lines. Endogenous PHB was detected by western blot and immunocytochemistry. Reactive oxygen species were determined by 5‐(and‐6)‐chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate fluorescence assay, and mitochondrial activity was assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT) reduction, uncoupling protein 2 expression and ATP production. Cell death was determined by Hoechst 33342 staining, cleaved caspase‐3 levels and flow cytometry. PHB was expressed in β‐cells under normal conditions and colocalized with Hoechst 33342 in the nucleus and with the mitochondrial probe Mitofluor in the perinuclear area. In ethanol‐treated cells, MTT reduction and ATP production decreased, whereas reactive oxygen species, uncoupling protein 2 and cleaved caspase‐3 levels increased. In addition, flow cytometry analysis showed an increase of apoptotic cells. Ethanol treatment increased PHB expression and induced PHB translocation from the nucleus to the mitochondria. PHB overexpression decreased the apoptotic effects of ethanol, whereas PHB knockdown enhanced these effects. The protective effects of endogenous PHB were recapitulated by incubation of the cells with recombinant human PHB. Thus, PHB is expressed in β‐cells, increases with oxidative stress and protects the cells against deleterious effects of ethanol.

Prohibitin interacts with phosphatidylinositol 3,4,5-triphosphate (PIP3) and modulates insulin signaling

Mitochondrial protein prohibitin (PHB) is known to associate with the plasma membrane of various cell types. However, biological function of plasma membrane associated PHB is not known. Recently we have shown that PHB undergoes tyrosine phosphorylation in response to insulin and here we report that PHB interacts with phosphatidylinositol 3,4,5-triphosphate (PIP3). Furthermore, we demonstrate that over expression of PHB attenuates insulin signaling downstream of phosphatidylinositol 3 (PI3) kinase. This effect was not observed with over expression of tyrosine phosphorylation site mutant-PHB suggesting a role for tyrosine phosphorylation of PHB in this process. Interestingly phosphorylation of PHB by Akt attenuates its interaction with PIP3 and enhances insulin signaling. Thus, phosphorylation of PHB and its interaction with PIP3 may be a part of the regulatory mechanisms that is involved in the modulation of insulin signaling. We speculate that phosphorylation of PHB may serve as a general mechanism in the regulation of PI3 kinase signaling including growth factors and immune receptor signaling.

Interaction between O-GlcNAc modification and tyrosine phosphorylation of prohibitin: implication for a novel binary switch.

Prohibitin (PHB or PHB1) is an evolutionarily conserved, multifunctional protein which is present in various cellular compartments including the plasma membrane. However, mechanisms involved in various functions of PHB are not fully explored yet. Here we report for the first time that PHB interacts with O-linked β-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and is O-GlcNAc modified; and also undergoes tyrosine phosphorylation in response to insulin. Tyrosine 114 (Tyr114) and tyrosine 259 (Tyr259) in PHB are in the close proximity of potential O-GlcNAc sites serine 121 (Ser121) and threonine 258 (Thr258) respectively. Substitution of Tyr114 and Tyr259 residues in PHB with phenylalanine by site-directed mutagenesis results in reduced tyrosine phosphorylation as well as reduced O-GlcNAc modification of PHB. Surprisingly, this also resulted in enhanced tyrosine phosphorylation and activity of OGT. This is attributed to the presence of similar tyrosine motifs in PHB and OGT. Substitution of Ser121 and Thr258 with alanine and isoleucine respectively resulted in attenuation of O-GlcNAc modification and increased tyrosine phosphorylation of PHB suggesting an association between these two dynamic modifications. Sequence analysis of O-GlcNAc modified proteins having known O-GlcNAc modification site(s) or known tyrosine phosphorylation site(s) revealed a strong potential association between these two posttranslational modifications in various proteins. We speculate that O-GlcNAc modification and tyrosine phosphorylation of PHB play an important role in tyrosine kinase signaling pathways including insulin, growth factors and immune receptors signaling. In addition, we propose that O-GlcNAc modification and tyrosine phosphorylation is a novel previously unidentified binary switch which may provide new mechanistic insights into cell signaling pathways and is open for direct experimental examination.