Immunophilins and the nervous system.

The search for immunosuppressant drugs to increase the success of organ transplantation led to the discovery of the immunophilins, proteins that interface with a range of signal transduction systems inside cells, especially in the nervous and immune systems. Here we review how these interesting molecules work and consider their therapeutic potential.

Interaction of RAFT1 with gephyrin required for rapamycin-sensitive signaling.

RAFT1 (rapamycin and FKBP12 target 1; also called FRAP or mTOR) is a member of the ATM (ataxia telangiectasia mutated)-related family of proteins and functions as the in vivo mediator of the effects of the immunosuppressant rapamycin and as an important regulator of messenger RNA translation. In mammalian cells RAFT1 interacted with gephyrin, a widely expressed protein necessary for the clustering of glycine receptors at the cell membrane of neurons. RAFT1 mutants that could not associate with gephyrin failed to signal to downstream molecules, including the p70 ribosomal S6 kinase and the eIF-4E binding protein, 4E-BP1. The interaction with gephyrin ascribes a function to the large amino-terminal region of an ATM-related protein and reveals a role in signal transduction for the clustering protein gephyrin.

Lymphocyte apoptosis: mediation by increased type 3 inositol 1,4,5-trisphosphate receptor.

B and T lymphocytes undergoing apoptosis in response to anti-immunoglobulin M antibodies and dexamethasone, respectively, were found to have increased amounts of messenger RNA for the inositol 1,4,5-trisphosphate receptor (IP3R) and increased amounts of IP3R protein. Immunohistochemical analysis revealed that the augmented receptor population was localized to the plasma membrane. Type 3 IP3R (IP3R3) was selectively increased during apoptosis, with no enhancement of type 1 IP3R (IP3R1). Expression of IP3R3 antisense constructs in S49 T cells blocked dexamethasone-induced apoptosis, whereas IP3R3 sense, IP3R1 sense, or IP3R1 antisense control constructs did not block cell death. Thus, the increases in IP3R3 may be causally related to apoptosis.

RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway.

Removal of damaged mitochondria is orchestrated by a pathway involving the PINK1 kinase and the PARKIN ubiquitin ligase. Ubiquitin chains assembled by PARKIN on the mitochondrial outer membrane recruit autophagy cargo receptors in complexes with TBK1 protein kinase. While TBK1 is known to phosphorylate cargo receptors to promote ubiquitin binding, it is unknown whether TBK1 phosphorylates other proteins to promote mitophagy. Using global quantitative proteomics, we identified S72 in RAB7A, a RAB previously linked with mitophagy, as a dynamic target of TBK1 upon mitochondrial depolarization. TBK1 directly phosphorylates RAB7AS72, but not several other RABs known to be phosphorylated on the homologous residue by LRRK2, in vitro, and this modification requires PARKIN activity in vivo. Interaction proteomics using nonphosphorylatable and phosphomimetic RAB7A mutants revealed loss of association of RAB7AS72E with RAB GDP dissociation inhibitor and increased association with the DENN domain-containing heterodimer FLCN-FNIP1. FLCN-FNIP1 is recruited to damaged mitochondria, and this process is inhibited in cells expressing RAB7AS72A. Moreover, nonphosphorylatable RAB7A failed to support efficient mitophagy, as well as recruitment of ATG9A-positive vesicles to damaged mitochondria. These data reveal a novel function for TBK1 in mitophagy, which parallels that of LRRK2-mediated phosphorylation of the homologous site in distinct RABs to control membrane trafficking.

Stress and mTORture signaling.

The TOR (target of rapamycin) pathway is an evolutionarily conserved signaling module regulating cell growth (accumulation of mass) in response to a variety of environmental cues such as nutrient availability, hypoxia, DNA damage and osmotic stress. Its pivotal role in cellular and organismal homeostasis is reflected in the fact that unrestrained signaling activity in mammals is associated with the occurrence of disease states including inflammation, cancer and diabetes. The existence of TOR homologs in unicellular organisms whose growth is affected by environmental factors, such as temperature, nutrients and osmolarity, suggests an ancient role for the TOR signaling network in the surveillance of stress conditions. Here, we will summarize recent advances in the TOR signaling field with special emphasis on how stress conditions impinge on insulin/insulin-like growth factor signaling/TOR signaling.

Neural actions of immunophilin ligands.

Immunophilins, protein receptors for immunosuppressant drugs such as cyclosporin A and FK506, are enriched far more in the brain than in the immune system. Drug-immunophilin complexes bind to calcineurin, inhibiting its phosphatase activity and leading to immunosuppressant effects. The immunophilin FKBP-12 (FK506 binding protein, 12 kDa) forms a complex with the ryanodine and inositol (1,4,5) trisphosphate (IP3) receptors to regulate their physiological release of intracellular Ca2+. Here, Solomon Snyder and colleagues describe how non-immunosuppressant as well as immunosuppressant immunophilin ligands are neurotrophic for numerous classes of damaged neurones, both in culture systems and intact animals. Their ability to stimulate functional regrowth of damaged sciatic, cortical cholinergic, dopamine and 5-HT neurones may have therapeutic relevance.

Raptor and mTOR: subunits of a nutrient-sensitive complex.

mTOR/RAFT1/FRAP is the target of the FKBP12-rapamycin complex as well as a central component of a nutrient- and hormone-sensitive pathway that controls cellular growth. Recent work reveals that mTOR interacts with a novel evolutionarily conserved protein that we named raptor, for "regulatory associated protein of mTOR." Raptor has several roles in the mTOR pathway. It is necessary for nutrient-mediated activation of the downstream effector S6K1 and increases in cell size. In addition, under conditions that repress the mTOR pathway, the association of raptor with mTOR is strengthened, leading to a decrease in mTOR kinase activity. Raptor is a critical component of the mTOR pathway that regulates cell growth in response to nutrient levels by associating with mTOR.

Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel autophagy regulator by high content shRNA screening.

Deregulation of autophagy has been linked to multiple degenerative diseases and cancer, thus the identification of novel autophagy regulators for potential therapeutic intervention is important. To meet this need, we developed a high content image-based short hairpin RNA screen monitoring levels of the autophagy substrate p62/SQSTM1. We identified 186 genes whose loss caused p62 accumulation indicative of autophagy blockade, and 67 genes whose loss enhanced p62 elimination indicative of autophagy stimulation. One putative autophagy stimulator, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), drives flux through pentose phosphate pathway. Knockdown of PFKFB4 in prostate cancer cells increased p62 and reactive oxygen species (ROS), but surprisingly increased autophagic flux. Addition of the ROS scavenger N-acetyl cysteine prevented p62 accumulation in PFKFB4-depleted cells, suggesting that the upregulation of p62 and autophagy was a response to oxidative stress caused by PFKFB4 elimination. Thus, PFKFB4 suppresses oxidative stress and p62 accumulation, without which autophagy is stimulated likely as a ROS detoxification response.

High-sensitivity sequencing of large proteins: partial structure of the rapamycin-FKBP12 target.

We report on studies leading to refinements of various steps of the protein internal sequencing process. Specifically, the developments comprise (1) higher-sensitivity chemical sequencing through background reduction; (2) improved peptide recovery from rapid in situ digests of nanogram amount, nitrocellulose-bound proteins; and (3) accurate UV spectroscopic identification of Trp- and Cys-containing peptides. In addition, we describe strategies for 2-dimensional liquid chromatographic peptide isolation from complex mixtures and a multi-analytical approach to peptide sequence analysis (Edman sequencing, matrix-assisted laser desorption mass spectrometry, and UV spectroscopy). Both strategies were applied in tandem to the primary structural analysis of a gel-purified, 250-kDa protein (mammalian target of rapamycin-FKBP12 complex), available in low picomolar quantities only. More than 300-amino acids worth of sequence was obtained in mostly uninterrupted stretches, several containing Trp, Cys, His, and Ser. That information has allowed the matching of a biological function of a mammalian protein to a yeast gene product with a well-characterized mutant phenotype. The results also demonstrate that extended chemical sequencing analysis (e.g., 26 successive amino acids) is now feasible, starting with initial yields well below 1 pmol.

Neural roles of immunophilins and their ligands.

The immunophilins are a family of proteins that are receptors for immunosuppressant drugs, such as cyclosporin A, FK506, and rapamycin. They occur in two classes, the FK506-binding proteins (FKBPs), which bind FK506 and rapamycin, and the cyclophilins, which bind cyclosporin A. Immunosuppressant actions of cyclosporin A and FK506 derive from the drug-immunophilin complex binding to and inhibiting the phosphatase calcineurin. Rapamycin binds to FKBP and the complex binds to Rapamycin And FKBP-12 Target (RAFT). RAFT affects protein translation by phosphorylating p70-S6 kinase, which phosphorylates the ribosomal S6 protein, and 4E-BP1, a repressor of protein translation initiation. Immunophilin levels are much higher in the brain than in immune tissues, and levels of FKBP12 increase in regenerating neurons in parallel with GAP-43. Immunophilin ligands, including nonimmunosuppressants that do not inhibit calcineurin, stimulate regrowth of damaged peripheral and central neurons, including dopamine, serotonin, and cholinergic neurons in intact animals. FKPB12 is physiologically associated with the ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors and regulates their calcium flux. By influencing phosphorylation of neuronal nitric oxide synthase, FKBP12 regulates nitric oxide formation, which is reduced by FK506.

Microarrays of cells expressing defined cDNAs.

Genome and expressed sequence tag projects are rapidly cataloguing and cloning the genes of higher organisms, including humans. An emerging challenge is to rapidly uncover the functions of genes and to identify gene products with desired properties. We have developed a microarray-driven gene expression system for the functional analysis of many gene products in parallel. Mammalian cells are cultured on a glass slide printed in defined locations with different DNAs. Cells growing on the printed areas take up the DNA, creating spots of localized transfection within a lawn of non-transfected cells. By printing sets of complementary DNAs cloned in expression vectors, we make microarrays whose features are clusters of live cells that express a defined cDNA at each location. Here we demonstrate two uses for our approach: as an alternative to protein microarrays for the identification of drug targets, and as an expression cloning system for the discovery of gene products that alter cellular physiology. By screening transfected cell microarrays expressing 192 different cDNAs, we identified proteins involved in tyrosine kinase signalling, apoptosis and cell adhesion, and with distinct subcellular distributions.

RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs.

The immunosuppressants rapamycin and FK506 bind to the same intracellular protein, the immunophilin FKBP12. The FKB12-FK506 complex interacts with and inhibits the Ca(2+)-activated protein phosphatase calcineurin. The target of the FKBP12-rapamycin complex has not yet been identified. We report that a protein complex containing 245 kDa and 35 kDa components, designated rapamycin and FKBP12 targets 1 and 2 (RAFT1 and RAFT2), interacts with FKBP12 in a rapamycin-dependent manner. Sequences (330 amino acids total) of tryptic peptides derived from the 245 kDa RAFT1 reveal striking homologies to the yeast TOR gene products, which were originally identified by mutations that confer rapamycin resistance in yeast. A RAFT1 cDNA was obtained and found to encode a 289 kDa protein (2549 amino acids) that is 43% and 39% identical to TOR2 and TOR1, respectively. We propose that RAFT1 is the direct target of FKBP12-rapamycin and a mammalian homolog of the TOR proteins.

Colony stimulating factor-1 (CSF-1) stimulates temperature dependent phosphorylation and activation of the RAF-1 proto-oncogene product.

The serine/threonine kinase RAF-1 is phosphorylated in intact macrophages in response to CSF-1 at 37 degrees C The augmented phosphorylation of RAF-1 and a concomitant increase in RAF-1 associated serine/threonine kinase activity are kinetically later events than CSF-1 induced protein tyrosine phosphorylation. Furthermore, phosphoamino acid analysis of RAF-1 reveals the presence of phosphoserine, trace amounts of phosphothreonine but no phosphotyrosine and the phosphorylated RAF-1 does not react with anti-phosphotyrosine antibodies. In contrast to CSF-1 induced protein tyrosine phosphorylation, RAF-1 phosphorylation and activation are temperature dependent and do not occur at 4 degrees C. Furthermore, coprecipitation experiments failed to reveal any noncovalent association of RAF-1 with the CSF-1 receptor. Thus, while RAF-1 is not a direct substrate for the CSF-1 receptor tyrosine kinase in vivo, its temperature dependent phosphorylation and activation represent an intriguing aspect of the CSF-1 response.

The rapamycin and FKBP12 target (RAFT) displays phosphatidylinositol 4-kinase activity.

The immunosuppressant rapamycin prevents cell cycle progression in several mammalian cell lines and the yeast Saccharomyces cerevisiae. In mammalian cells, rapamycin binds to the small FK506-binding protein, FKBP12, allowing the drug-receptor complex to interact with the 289-kDa RAFT1/FRAP proteins. These proteins, along with their yeast homologs, TOR1/DRR1 and TOR2/DRR2, contain a C-terminal domain with amino acid homology to several phosphatidylinositol (PI) 4- and 3-kinases. However, no direct demonstration of kinase activity for this family of proteins has been reported. We now show that RAFT1, immunoprecipitated from rat brain and MG63 and HEK293 cells, contains PI 4-kinase activity and that rapamycin-FKBP12 has no effect on this activity. Thus, it is likely that, in vivo, rapamycin does not directly inhibit the PI 4-kinase activity and affects the RAFT1/FRAP protein through another mechanism.

Immunophilin FK506 binding protein associated with inositol 1,4,5-trisphosphate receptor modulates calcium flux.

The immunophilin FK506 binding protein 12 (FKBP12) is associated with and modulates the ryanodine receptor calcium release channel of skeletal muscle. Ryanodine receptor has amino acid homology and functional similarity with another intracellular Ca2+ release channel, the inositol 1,4,5-trisphosphate receptor (IP3R). In the present study we show that highly purified preparations of IP3R contain FKBP12. The complex of these two proteins is disrupted by the immunosuppressants FK506 and rapamycin, both of which are known to bind FKBP12 with high affinity. Disrupting the IP3R-FKBP12 interaction increases Ca2+ flux through IP3R, an effect that is reversed by added FKBP12. FKBP12 appears to be physiologically linked to IP3R, regulating its Ca2+ conductance.

RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1.

The complex of rapamycin with its intracellular receptor, FKBP12, interacts with RAFT1/FRAP/mTOR, the in vivo rapamycin-sensitive target and a member of the ataxia telangiectasia mutated (ATM)-related family of kinases that share homology with the catalytic domain of phosphatidylinositol 3-kinase. The function of RAFT1 in the rapamycin-sensitive pathway and its connection to downstream components of the pathway, such as p70 S6 kinase and 4E-BP1, are poorly understood. Here, we show that RAFT1 directly phosphorylates p70(S6k), 4E-BP1, and 4E-BP2 and that serum stimulates RAFT1 kinase activity with kinetics similar to those of p70(S6k) and 4E-BP1 phosphorylation. RAFT1 phosphorylates p70(S6k) on Thr-389, a residue whose phosphorylation is rapamycin-sensitive in vivo and necessary for S6 kinase activity. RAFT1 phosphorylation of 4E-BP1 on Thr-36 and Thr-45 blocks its association with the cap-binding protein, eIF-4E, in vitro, and phosphorylation of Thr-45 seems to be the major regulator of the 4E-BP1-eIF-4E interaction in vivo. RAFT1 phosphorylates p70(S6k) much more effectively than 4E-BP1, and the phosphorylation sites on the two proteins show little homology. This raises the possibility that, in vivo, an unidentified kinase analogous to p70(S6k) is activated by RAFT1 phosphorylation and acts at the rapamycin-sensitive phosphorylation sites of 4E-BP1.

The 12 kD FK 506 binding protein FKBP12 is released in the male reproductive tract and stimulates sperm motility.

BACKGROUND: The 12 kD FK506 binding protein FKBP12 is a cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin. In addition to its critical role in drug-induced T-cell immunosuppression, FKBP12 associates physiologically with ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors, regulating their ability to flux calcium. We investigated a role for FKBP12 in male reproductive physiology on the basis of our identification of extremely high levels of [3H]FK506 binding in male reproductive tissues. MATERIALS AND METHODS: [3H]FK506 binding studies were performed to identify tissues enriched with FK506 binding sites. The abundant [3H]FK506 binding sites identified in the male reproductive tract were localized by [3H]FK506 autoradiography. FK506 affinity chromatography was employed to purify FKBP from epididymal fluid. Anti-FKBP12 Western analysis was used to confirm the identity of the purified FKBP. The binding of exogenous FKBP12 to sperm was evaluated by [32P]FKBP12 binding studies and [33P]FKBP12 autoradiography. The effect of recombinant FKBP12 on sperm motility was investigated using a Hamilton Thorne motility analyzer. RESULTS: Male reproductive tissues contained high levels of [3H]FK506 binding. The localization of [3H]FK506 binding sites to the tubular epithelium of the caput epididymis and the lumen of the cauda and vas deferens suggested that FKBP is released in the male reproductive tract. FKBP12 was purified from epididymal plasma by FK506 affinity chromatography. Radiolabeled FKBP12 specifically bound to immature but not mature sperm. In sperm motility studies, FKBP12-treated caput sperm exhibited double the curvilinear velocity of untreated controls. CONCLUSIONS: High levels of FKBP12 are released in the male reproductive tract and specifically associate with maturing sperm. Recombinant FKBP12 enhances the curvilinear velocity of immature sperm, suggesting a role for FKBP12 in motility initiation. The highest concentrations of soluble FKBP12 in the male reproductive tract occur in the lumen of the vas deferens, a site of sperm storage and the conduit for ejaculated sperm. Preservation of mammalian sperm for reproductive technologies may be optimized by supplementing incubation or storage media with FKBP12.

Neurabin is a synaptic protein linking p70 S6 kinase and the neuronal cytoskeleton.

p70 S6 kinase (p70(S6k)) is a mitogen-activated protein kinase that plays a central role in the control of mRNA translation. It physiologically phosphorylates the S6 protein of the 40s ribosomal subunit in response to mitogenic stimuli and is a downstream component of the rapamycin-sensitive pathway, which includes the 12-kDa FK506 binding protein and includes rapamycin and the 12-kDa FK506 binding protein target 1. Here, we report the identification of neurabin (neural tissue-specific F-actin binding protein), a neuronally enriched protein of 1,095 amino acids that contains a PDZ domain and binds p70(S6k). We demonstrate the neurabin-p70(S6k) interaction by yeast two-hybrid analysis and biochemical techniques. p70(S6k) and neurabin coimmunoprecipitate from transfected HEK293 cells. Site-directed mutagenesis of neurabin implicates its PDZ domain in the interaction with p70(S6k), and deletion of the carboxyl-terminal five amino acids of p70(S6k) abrogates the interaction. Cotransfection of neurabin in HEK293 cells activates p70(S6k) kinase activity. The mRNA of neurabin and p70(S6k) show striking colocalization in brain sections by in situ hybridization. Subcellular fractionation of rat brain demonstrates that neurabin and p70(S6k) both localize to the soluble fraction of synaptosomes. By way of its PDZ domain, the neuronal-specific neurabin may target p70(S6k) to nerve terminals.