FK506 is a macrolide produced by fungi and used in medicine as an immuno-suppressant to prevent rejection of organs following transplantation. A number of proteins have been identified that bind this drug. FK506-binding proteins (FKBPs) constitute a large family of enzymes found in all classes of organisms, including plants. These enzymes are peptidyl-prolyl cis-trans isomerases (PPiases) that catalyze the interconversion of the peptide bond on the amino terminal side of proline from the cis configuration to the trans configuration. This interconversion impacts protein structure, because proline in the cis configuration causes kinks within the molecule. FKBPs are also known to participate in many cellular processes such as cell signaling, protein transport and transcription. FKBPs associate with many other proteins to form complexes, but the connections between FKBP foldase activity, interactions with other protein partners, and impact on growth and developmental processes are only beginning to be understood.
Plant FKBPs have recently been identified, and disruption of the genes encoding these proteins has indicated that they play an important role in the regulation of cell division and differentiation. In a review article in the September 2001 issue of Trends in Plant Science (6:426-431), Yaël Harrar, Catherine Bellini and Jean-Denis Faure (Laboratoire de Biologie Cellulaire, INRA, France) discuss the current status of knowledge on the structure and functions of FKBPs. The authors initially focus on FKBP research in mammals, then extend the discussion to studies on plant FKBPs.
FKBP isoforms fall into two general categories, small FKBPs and large FKBPs. One small animal FKBP isoform, FKBP12, is involved in immunosuppression. When associated with immunosuppressive drugs such as FK506 or rapamycin, FKBP12 inhibits T-cell activation. Both FK506 and rapamycin can bind FKBP12, but the resulting immunosuppression occurs by different mechanisms for each respective drug. Study of the mechanisms of FK506 and rapamycin action in mammalian systems has allowed the clarification of signal transduction pathways for lymphocyte activation. In absence of these drugs, FKBP12 normally associates with receptors such as TGF-beta receptor type II or calcium channels such as the ryanodine receptor or the inositol-(1,4,5)-triphosphate receptor. These channels are regulated indirectly by FK506 through its interaction with negative regulators of channel receptors.
Various model systems have been used to study FKBP functions. Yeast studies have not been as fruitful as hoped, as disruption of every FKBP and cyclophilin in a single yeast strain did not cause any effect on cell survival. Disruption of one additional gene encoding an enzyme with PPiase activity resulted in lethality. In mice, a knockout line of FKBP12 showed cardiac defect and early death. This result is consistent with a previously suggested role of FKBP12 in cardiac calcium channel complex regulation.
FKBPs of high molecular weights have additional domains not found in the smaller FKBPs. In general, large FKPBs contain one or more FKBP12-like domains, a tetratricopeptide repeat (TPR), and a C-terminal domain that may bind calmodulin. FKBP51 and FKBP52 are the most well-characterized large FKBPs. Both of these proteins associate with hsp90 (heat shock protein 90) through their TPR domain in the native steroid receptor complex. Hsp90 is a molecular chaperone, and FKBP52 itself has shown molecular chaperone activity. At least two other proteins present in the same complex are also molecular chaperones, and it has been suggested that this complex has a major role in directing protein folding in the cytosol. Furthermore, FKBP52 may associate with dynein, a protein that regulates movement along microtubules. It is possible that the interaction of the chaperone complex with dynein through FKBP52 may allow the steroid receptor to translocate along the cytoskeleton, though this remains to be confirmed.
FKBPs have also been identified in plants, first reported in Vicia faba, wheat and Arabidopsis. Vicia faba VfFKBP13, which is similar to mammalian FKBP13, was purified by its ability to bind FK506. This enzyme does show PPiase activity. VfFKBP13 contains a region of hydrophobic amino acids characteristic of a signal peptide for localization to the ER. Knowledge of the VfFKBP13 sequence allowed identification of the Arabidopsis orthologs AtFKBP15-1 and AtFKBP15-2. Large isoforms of FKBPs have also been identified, such as wheat wFKBP73, which shows similarity to FKBP52. The wFKBP73 protein also binds FK506 and has PPiase activity. Other large FKBPs have subsequently been identified in Arabidopsis, maize and wheat.
The sequencing of the Arabidopsis genome allowed identification of 17 FKBPs and FKBP-like proteins. Of these, 11 are small FKBPs and six are large FKBPs. Phylogenetic analyses using FKBP12-like domains for comparison of plant and animal FKBPs indicate that some plant FKBPs are closely related to mammalian FKBPs, while others are more distantly related and may serve plant-specific roles. Indeed, some studies have indicated that certain plant FKBPs are not interchangeable with mammalian FKBPs. For example, Vicia FKBP12 could not complement the corresponding yeast mutant, nor could it bind the interacting protein calcineurin in vitro when associated with FK506, whereas AtFKPB12 may act as a functional equivalent of FKBP12. This suggests that the functional roles of VfFKBP12 and AtFKPB12 have diverged considerably.
Several of the large Arabidopsis FKBPs have been studied in some detail. Gene expression studies indicate that two members of this family, TWD and PAS1, are expressed constitutively although PAS1 is up-regulated by cytokinins. Other members of the large molecular weight FKBP family in Arabidopsis show expression regulation in response to stresses such as wounding, salt or heat shock.
Transgenic plants have been generated to understand the function of large wFKBPs from wheat. Transgenic rice plants overexpressing full-length wFKBP73 resulted in fertile plants, while expression of a truncated version of wFKBP73 caused male sterility. Transgenic wheat plants with altered levels of wFKBP77 have also been produced. Overexpression of wFKBP77 caused abnormal morphological states such as dwarfism, altered leaf shape and sterility. This suggests that accumulation of FKBP adversely affects plant development.
Gene disruptions further support a role for FKBPs in plant development. The pas1 (PASTICCINO1) mutant of Arabidopsis shows ectopic cell division and cell differentiation defects. In the presence of cytokinins the phenotype is enhanced to result in plants that resemble green calli. Seedlings of pas1 mutants have misshapened cotyledons that never expand, among other developmental abnormalities, and few individuals are able to develop flowers. A second Arabidopsis FKBP mutant is twisted dwarf (twd), which has shortened organs and a twisted growth phenotype. The TWD gene encodes a 42-kDa FKBP-like protein that may be associated with the cell membrane. Genetic studies with twd and mutants affected in brassinolide biosynthesis show that TWD1 is essential for perception of brassinolide.
In conclusion, the authors note that PCR-based reverse genetic screens of T-DNA and transposon-tagged Arabidopsis populations should allow the identification of more FKBP mutants in the near future. This, in turn, should facilitate the functional characterization of members of this gene family.