The reversible protein phosphorylation on serine or threonine residues that precede proline (pSer/Thr-Pro) is a key signaling mechanism for the control of various cellular processes, including cell division. The pSer/Thr-Pro moiety in peptides exists in the two completely distinct cis and trans conformations whose conversion is catalyzed specifically by the essential prolyl isomerase Pin1. Previous results suggest that Pin1 might regulate the conformation and dephosphorylation of its substrates. However, it is not known whether phosphorylation-dependent prolyl isomerization occurs in a native protein and/or affects dephosphorylation of pSer/Thr-Pro motifs. Here we show that the major Pro-directed phosphatase PP2A is conformation-specific and effectively dephosphorylates only the trans pSer/Thr-Pro isomer. Furthermore, Pin1 catalyzes prolyl isomerization of specific pSer/Thr-Pro motifs both in Cdc25C and tau to facilitate their dephosphorylation by PP2A. Moreover, Pin1 and PP2A show reciprocal genetic interactions, and prolyl isomerase activity of Pin1 is essential for cell division in vivo. Thus, phosphorylation-specific prolyl isomerization catalyzed by Pin1 is a novel mechanism essential for regulating dephosphorylation of certain pSer/Thr-Pro motifs.
The phosphorylation-specific peptidyl prolyl cis/trans isomerase (PPIase) Pin1 in humans and its homologues in yeast and animal species play an important role in cell cycle regulation. These PPIases consist of an NH 2 -terminal WW domain that binds to specific phosphoserine-or phosphothreonine-proline motifs present in a subset of phosphoproteins and a COOH-terminal PPIase domain that specifically isomerizes the phosphorylated serine/threonine-proline peptide bonds. Here, we describe the isolation of MdPin1, a Pin1 homologue from the plant species apple (Malus domestica) and show that it has the same phosphorylation-specific substrate specificity and can be inhibited by juglone in vitro, as is the case for Pin1. A search in the plant expressed sequence tag data bases reveals that the Pin1-type PPIases are present in various plants, and there are multiple genes in one organism, such as soybean (Glycine max) and tomato (Lycopersicon esculentum). Furthermore, all these plant Pin1-type PPIases, including AtPin1 in Arabidopsis thaliana, do not have a WW domain, but all contain a four-amino acid insertion next to the phosphospecific recognition site of the active site. Interestingly, like Pin1, both MdPin1 and AtPin1 are able to rescue the lethal mitotic phenotype of a temperature-sensitive mutation in the Pin1 homologue ESS1/PTF1 gene in Saccharomyces cerevisiae. However, deleting the extra four amino acid residues abolished the ability of AtPin1 to rescue the yeast mutation under non-overexpression conditions, indicating that these extra amino acids may be important for mediating the substrate interaction of plant enzymes. Finally, expression of MdPin1 is tightly associated with cell division both during apple fruit development in vivo and during cell cultures in vitro. These results have demonstrated that phosphorylationspecific PPIases are highly conserved functionally in yeast, animal, and plant species. Furthermore, the experiments suggest that although plant Pin1-type enzymes do not have a WW domain, they may fulfill the same functions as Pin1 and its homologues do in other organisms.
Material and methods Cloning of a full-length eDNA for hCyP33PCR amplification of DNA-fragments from different human Jurkat T cell line cDNA libraries were performed with two degenerate primers, which correspond to amino acid sequences QGGDF and KHVVFG (boxed in Fig. 2b) in the highly conserved regions of known cyclophilins [3,13,[17][18][19][20]. The resulting PCR products were subcloned into pUC18 and sequenced. Nested PCR was performed to extend the sequence using two libraries: a Marathon cDNA library of the human Jurkat T cell line (Clontech) and a cDNA library made of mRNA from human T cells. Both libraries yielded as longest cDNA one with a length of 1.6 kb. The eDNA was sequenced on both strands.
The codon usage of a hybrid bacterial gene encoding a thermostable (1,3-1,4)-13-glucanase was modified to match that of the barley (1,3-1,4)-p-glucanase isoenzyme (4) and their degradation is a prerequisite for the enzymatic mobilization of endosperm storage components, which serve as nutrients for the growing embryo. Efficient degradation of endosperm cell walls is also important for utilization of barley as a monogastric animal feed (5, 6) and in industrial processes such as malting and brewing (7). Furthermore, extraction of non-food products deposited in the endosperm of transgenic barley would be facilitated by the action of highly efficient, heat stable cell wall-degrading enzymes. The (1,3-1,4)-3-glucanases (EC 3.2
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