Phosphorylation of proteins on serine͞threonine residues preceding proline is a key signaling mechanism. The conformation and function of a subset of these phosphorylated proteins is regulated by the prolyl isomerase Pin1 through isomerization of phosphorylated Ser͞Thr-Pro bonds. Although young Pin1 ؊/؊ mice have been previously shown to develop normally, we show here that they displayed a range of cell-proliferative abnormalities, including decreased body weight and testicular and retinal atrophies. Furthermore, in Pin1 ؊/؊ adult females, the breast epithelial compartment failed to undergo the massive proliferative changes associated with pregnancy. Interestingly, many of these Pin1-deficient phenotypes such as retinal hypoplasia and mammary gland impairment are also the characteristic of cyclin D1-deficient mice. Cyclin D1 levels were significantly reduced in many tissues in Pin1-deficient mice, including retina and breast epithelial cells from pregnant mice. Moreover, Pin1 directly bound to cyclin D1 phosphorylated on Thr-286 -Pro increased cyclin D1 in the nucleus and stabilized cyclin D1. These results indicate that Pin1 positively regulates cyclin D1 function at the transcriptional level, as demonstrated previously, and also through posttranslational stabilization, which together explain why Pin1 loss-of-function phenotypes in the mouse resemble cyclin D1-null phenotypes. Our results provide genetic evidence for an essential role of Pin1 in maintaining cell proliferation and regulating cyclin D1 function. P hosphorylation of proteins on serine͞threonine residues preceding proline (pSer͞Thr-Pro) is a key regulatory mechanism for the control of various cellular processes, including cell division and transcription (for reviews see refs. 1-3). The pSer͞Thr-Pro moiety in peptides and proteins exists in two distinct cis and trans conformations, whose conversion is catalyzed specifically by Pin1 (4, 5). Pin1 is a cis͞trans peptidyl-prolyl isomerase that acts only on phosphorylated Ser͞Thr-Pro bonds (6-8). In addition, Pin1 contains an N-terminal WW domain, which functions as a phosphorylated Ser͞Thr-Pro binding module (9, 10). This phosphorylationdependent interaction targets Pin1 to a defined subset of phosphorylated substrates facilitating conformational changes in phosphorylated proteins, thereby regulating their biological function (7,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Thus, Pin1-dependent prolyl isomerization is an essential and novel postphosphorylation regulatory mechanism.Given its phosphorylated Ser͞Thr-Pro substrate specificity, Pin1 has also been shown to be essential for maximal cell growth in different systems (4, 5). Interestingly, we have recently found that Pin1 is strikingly overexpressed in most human breast cancer tissues (21,22). Pin1 levels are correlated with cyclin D1 mRNA and protein levels in human cancer tissues. Moreover, Pin1 can activate the cyclin D1 promoter in cell lines via binding phosphorylated c-Jun and ␤-catenin and increasing their transcriptional activity (21,22). The...

We have isolated and characterized two suppressor genes, SUI4 and SUI5, that can initiate translation in the absence of an AUG start codon at the HIS4 locus in Saccharomyces cerevisiae. Both suppressor genes are dominant in diploid cells and lethal in haploid cells. The SUI4 suppressor gene is identical to the GCD11 gene, which encodes the ␥ subunit of the eIF-2 complex and contains a mutation in the G 2 motif, one of the four signature motifs that characterizes this subunit to be a G-protein. The SUI5 suppressor gene is identical to the TIF5 gene that encodes eIF-5, a translation initiation factor known to stimulate the hydrolysis of GTP bound to eIF-2 as part of the 43S preinitiation complex. Purified mutant eIF-5 is more active in stimulating GTP hydrolysis in vitro than wild-type eIF-5, suggesting that an alteration of the hydrolysis rate of GTP bound to the 43S preinitiation complex during ribosomal scanning allows translation initiation at a non-AUG codon. Purified mutant eIF-2␥ complex is defective in ternary complex formation and this defect correlates with a higher rate of dissociation from charged initiator-tRNA in the absence of GTP hydrolysis. Biochemical characterization of SUI3 suppressor alleles that encode mutant forms of the ␤ subunit of eIF-2 revealed that these mutant eIF-2 complexes have a higher intrinsic rate of GTP hydrolysis, which is eIF-5 independent. All of these biochemical defects result in initiation at a UUG codon at the his4 gene in yeast. These studies in light of other analyses indicate that GTP hydrolysis that leads to dissociation of eIF-2 ⅐ GDP from the initiator-tRNA in the 43S preinitiation complex serves as a checkpoint for a 3-bp codon/anticodon interaction between the AUG start codon and the initiator-tRNA during the ribosomal scanning process.

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