The expression of cyclin-dependent kinase inhibitor p27 kip1 in human tumors and normal tissues was investigated using a panel of novel anti-p27 kip1 mAbs. An inverse correlation between expression of p27 kip1 and cell proliferation was generally observed after analyzing its expression in 25 different normal human tissues. In some highly proliferative human breast cancer cells, however, high level p27 kip1 expression was seen, indicating the existence of a mechanism by which some growing tumor cells may tolerate this inhibitor of cell cycle progression. Detailed studies demonstrated a correlation between the high level expression of p27 kip1 and cyclin D1 in human breast cancer cells. There was also an inverse correlation between the expression of p27 kip1 and the degree of tumor malignancy in human breast and colorectal cancers, indicating that p27 kip1 may be a useful prognostic marker in these cancers.

E2F1 overexpression has been shown to induce apoptosis in cooperation with p53. Using Saos-2 cells, which are null for p53 and lack functional Rb, we have demonstrated that E2F1 overexpression can also induce apoptosis in the absence of p53 and retinoblastoma protein (Rb). E2Fl-induced apoptosis can be specifically inhibited by Rb but not mdm2, which is known for its ability to inhibit p53-induced apoptosis. Through the study of the apoptotic function of a set of E2F1 mutants, it was clear that the transactivation and the apoptotic function of E2F1 are uncoupled. The transactivation-defective E2F1 mutants E2Fl(1-374), E2Fl(390-1)DF(Amdm2), and E2Fl(406-415)(ARb) can induce apoptosis as effectively as wild-type E2F1. In contrast to E2F1 transactivation, the DNA-binding activity of E2F1 was proven to be essential for its apoptotic function, as the DNA-binding-defective mutants E2Fl(132) and E2Fl(132)(1-374) failed to induce apoptosis. Therefore Rb may inhibit E2Fl-induced apoptosis by mechanisms other than the suppression of the transactivation of E2F1. This hypothesis was supported by our observation that although Rb overexpression can specifically repress the apoptosis induced by wild-type E2F1 and a Rb-binding-competent E2F1 mutant E2Fl(390-1)DF(Amdm2), it failed to inhibit the apoptosis induced by mutants E2Fl(1-374) and E2Fl(~406-415)(~Rb), which are defective or reduced in Rb binding and transactivation. All of these points argue for a novel function for E2F1 and Rb in controlling apoptosis. The results also indicate that transcriptional repression rather than the transactivation function of E2F1 may be involved in its apoptotic function. The results presented here may provide us some physiological implication of the repression function of the Rb-E2F1 complex.

Translocation of precursor proteins across the mitochondrial membranes requires the coordinated action of multisubunit translocases in the outer and inner membrane, and the driving force for translocation across the inner membrane is provided by the matrixlocated heat shock protein 70 (mtHsp70). The central components of the protein import machinery are essential. Here we describe Zim17, an essential protein with a zinc finger motif involved in protein import into mitochondria. Comparative genomics suggested a correction to the open reading frame of YNL310c, the gene encoding Zim17 in Saccharomyces cerevisiae. The revised open reading frame codes for a classic mitochondrial targeting signal, which is processed from Zim17 in the mitochondrial matrix. Loss of Zim17 selectively diminishes import of proteins into the matrix of mitochondria, but this loss of Zim17 is partially suppressed by overexpression of the J-protein Pam18/Tim14. We propose that Zim17 functions as an example of a "fractured" J-protein, where a protein like Zim17 contributes a zinc finger domain to Type III J-proteins, in toto providing for substrate loading onto Hsp70.Molecular chaperones of the 70-kDa heat shock protein (Hsp70) family bind short, unfolded, hydrophobic segments of substrate polypeptides (1). The Hsp70 has both a substratebinding domain and an ATPase domain, and substrate-binding activity is coupled to ATP hydrolysis (2, 3). A member of the J-protein family usually works as a coupling factor, stimulating ATPase activity of the Hsp70 once the Hsp70 has substrate occupying its substrate-binding domain (3). J-proteins are characterized by a small J-domain that docks to and stimulates the ATPase domain of Hsp70, and a combination of zinc finger and carboxyl-terminal domains that together assist binding non-native structures in a substrate polypeptide (4 -6). In combination, the J-protein can load substrate into the bindingpocket of Hsp70 and stimulate the hydrolysis of ATP for productive chaperone action.The import of mitochondrial proteins requires the activity of Hsp70s at several stages (7-9). Most mitochondrial proteins are synthesized in the cytosol, and these relatively hydrophobic polypeptides must remain unfolded (or be actively unfolded) to cross the mitochondrial membranes and only subsequently fold to their final structure (9 -11). In the later stages of the mitochondrial protein import pathway, Hsp70 activity is localized at the TIM23 complex (one of the translocases in the inner mitochondrial membrane) where cycles of ATP hydrolysis by the chaperone are used to effectively drive translocation of the substrate across the membrane. Precursor proteins encounter the mitochondrial Hsp70, mtHsp70 (also called Ssc1), immediately upon entry of the terminus of the unfolded polypeptide in the matrix (12, 13); mtHsp70 provides both the driving force for translocation of polypeptides through the TIM23 complex and works in the matrix to fold protein substrates once imported (9, 14).J-proteins in the mitochondrial matrix can regulate ...

No abstract