Transcriptional activation of target genes represents an important component of the tumour-suppressor function of p53 and provides a functional link between p53 and various growth-regulatory processes, including cell cycle progression (p21/WAF1), DNA repair (GADD45) and apoptosis (bax). Here we use a differential cloning approach to identify the gene encoding insulin-like growth factor binding protein 3 (IGF-BP3) as a novel p53-regulated target gene. Induction of IGF-BP3 gene expression by wild-type but not mutant p53 is associated with enhanced secretion of an active form of IGF-BP3 capable of inhibiting mitogenic signalling by the insulin-like growth factor IGF-1. Our results indicate that IGF-BP3 may link p53 to potential novel autocrine/paracrine signalling pathways and to processes regulated by or dependent on IGF(s), such as cellular growth, transformation and survival.

The p53 tumor suppressor protein is a transcription factor with sequence-specific DNA binding activity that is thought to be important for the growth-inhibitory function of p53. DNA binding appears to require activation of a cryptic form of p53 by allosteric mechanisms involving a negative regulatory domain at the carboxyl terminus of p53. The latent form of p53, reactive to the carboxyl-terminal antibody PAb421, is produced in a variety of eukaryotic cells, suggesting that activation of p53 is an important rate-limiting step in vivo. In this report we provide evidence that phosphorylation of serine 378 within the carboxyl-terminal negative regulatory domain of the human p53 protein by protein kinase C correlates with loss of PAb421 reactivity and a concomitant activation of sequence-specific DNA binding. These effects are reversed by subsequent dephosphorylation of the protein kinase C-reactive site by protein phosphatases 1 (PP1) and 2A (PP2A), which restore the reactivity of p53 to PAb421 and regenerate the latent form of p53 lacking significant DNA binding activity. Thus, p53 is subject to both positive and negative regulation by reversible enzymatic modifications affecting the latent or active state of the protein, suggesting a possible mechanism for the regulation of its tumor suppressor function.

A 15-mer phage display random peptide library was screened with purified bovine Hsc70, and nucleotide sequence analysis of the selected clones showed a large enrichment for peptides containing basic sequences with at least KK, KR, or RR. Binding affinity for Hsc70 of representative peptides increased dramatically for heptamers compared with hexamers. The peptide NIVRKKK had the highest affinity for Hsc70, and substitution analyses showed that hydrophobic residues followed by basic residues play important roles in maintaining this affinity. In contrast, NIVRKKK was a weaker stimulator of the Hsc70 ATPase activity compared with pigeon cytochrome c peptide and FYQLALT, a peptide optimized for binding to Hsc70. FYQLALT effectively blocked the binding of NIVRKKK to Hsc70, possibly by causing a conformational change that masked Hsc70's binding site for the basic peptide. Two hypotheses are offered to explain the two different peptide motifs. First, it is proposed that Hsc70 recognizes two different amino acid sequence motifs in its dual roles of chaperoning proteins to organelles (NIVRKKK-like sequences) and facilitating protein folding (FYQLALT-like sequences). Second, the NIVRKKK motif may be used to bind certain folded proteins with which Hsc70 interacts, such as itself, p53, and Dnaj2.

In this report we show that: (1) molecular chaperones in the heat shock protein (hsp) family are a new class of cellular proteins induced by Transforming Growth Factor-PI (TGFP), a cytokine present in serum, (2) rapid induction of Hsc70 precedes a general increase in protein synthesis and may be a preparatory event,(3) TGFP is a potent regulator of overall protein synthesis in chicken embryo cells (CEC), and (4) isoforms of tisp90 with different biochemical properties exist, raising the possibility that they may have different functions. TGFP can substit~ite for serum in stimulating synthesis of members ol the Hsp90 and Hsp70 families of stress proteins, whereas other cytokines, including PDGF, FGF, and EGF, were not effective nor did they enhance the stimulatory effect of TGFP on the hsp's. Analysis of the induction of hsp's using one-and two-dimensional polyacrylamide gel electrophore5is indicated that members of the Hsp70 family of molecular chaperones were induced rapidly by TGFP, reaching maximum rates of accumulation by 5 hours of treatment. Total protein synthesk increased more slowly, undergoing an -twofold increase in 24 hours. Using a modified protocol for two-dimensional gel electrophoresis, the Hsp90 protein family was separated into four isoelectric forms, two of which were phosphorylated (Hsp90-2 and -4). These phosphorylated isoforrns turned over faster than the unphosphorylated forms of HspY0. All four isoforms were heat inducible, but only Hsp90-2 and -3 were induced rapidly by TGFP, again within 5 hours of treatment. The effects oiserum on these protein families were similar to those of TGFP, suggesting that this cytokine may be the serum component primarily responsible for up-regulating members of the Hsp90 and Hsp70 families. We hypothesize that cells rapidly increase thcir chaperoning capacity for newly synlhesired polypeptides in preparation for an increase in the rate of synthesis of proteins up-regulated by TGFP.1992 Wiley-Lis5, Inc.

: In this study, we examined the expression and subcellular localization of cyclin‐dependent kinase 5 (Cdk5), cyclin D1, and cyclin E in Leydig and Sertoli cell lines that were cultured with 7.5, 1.0, 0.5, or 0% serum (mixture of a 2:1 ratio of horse serum and fetal bovine serum) and in the developing rat testis to verify the possible functions of Cdk5, cyclin D1, and cyclin E in the testis. The abundance of Cdk5 and cyclin E in the Leydig cell line, TM3, was significantly reduced at low serum concentrations. In contrast, serum concentration had no effect on Cdk5 and cyclin E levels in the Sertoli cell line, TM4. Cyclin D1 was detected by western blot analysis in TM4 cells only, and its abundance was serum dose dependent. The kinase activity of Cdk5 in TM3 and TM4 cells that were cultured at various serum concentrations coincided with the levels of Cdk5 expression. Immunohistochemical staining for Cdk5 and cyclin E reealed nuclear and cytoplasmic distribution, both in TM3 and TM4 cells. Moreover, cyclin D1 immunoreactivity was only detected in TM4 cells. In the developing rat testis, Cdk5 expression was most prominent at 2 and 3 weeks after birth. Cyclin D1 was strongly expressed at 1 and 2 weeks in premature rat testes. On the other hand, cyclin E was highly expressed in the adult testis. Immunohistochemical localization of Cdk5, cyclin D1, and cyclin E in 1‐week‐old and adult rat testes revealed expression in both Leydig and Sertoli cells. Our results suggest that Cdk5 in TM3 and Leydig cells of the testis might play a role in cell cycle regulation, whereas Cdk5 in TM4 and Sertoli cells of the adult testis might have some additional functions besides control of proliferation. Key words: Kinase activity, growth control.