No abstract

The 14-3-3 proteins have been implicated as potential regulators of diverse signaling pathways. Here, using two-hybrid assays and in vitro assays of protein interaction, we show that the ⑀ isoform of 14-3-3 interacts with the insulin-like growth factor I receptor (IGFIR) and with insulin receptor substrate I (IRS-1), but not with the insulin receptor (IR). Coprecipitation studies demonstrated an IGFI-dependent in vitro interaction between 14-3-3-glutathione S-transferase proteins and the IGFIR. In similar studies no interaction of 14-3-3 with the IR was observed. We present evidence to suggest that 14-3-3 interacts with phosphoserine residues within the COOH terminus of the IGFIR. Specifically, peptide competition studies combined with mutational analysis suggested that the 14-3-3 interaction was dependent upon phosphorylation of IGFIR serine residues 1272 and/or 1283, a region which has been implicated in IGFIRdependent transformation. Phosphorylation of these serines appears to be dependent upon prior IGFIR activation since no interaction of 14-3-3 was observed with a kinase-inactive IGFIR in the two-hybrid assay nor was any in vitro interaction with unstimulated IGFIR derived from mammalian cells. We show that the interaction of 14-3-3 with IRS-1 also appears to be phosphoserinedependent. Interestingly, 14-3-3 appears to interact with IRS-1 before and after hormonal stimulation. In summary, our data suggest that 14-3-3 interacts with phosphoserine residues within the COOH terminus of the IGFIR and within the central domain of IRS-1. The potential functional roles which 14-3-3 may play in IGFIR and IRS-1-mediated signaling remain to be elucidated.The insulin and IGFI 1 receptors share a high degree of homology (1), and both are believed to signal in part via the IRS and SHC proteins (2-6). Despite the apparently identical signaling events which are initiated by the IR and IGFIR via these signaling proteins, the primary physiological role of insulin is to regulate metabolic events, while IGFI primarily regulates cellular growth, differentiation, and transformation (7). It is therefore likely that divergent signaling pathways exist that are responsible for mediating these different cellular effects. Several studies have suggested the existence of such divergent pathways. First, receptor chimeras in which the cytoplasmic domain of the IGFIR was fused to the IR, were reported to have significantly increased mitogenic potential compared with the IR (8). Another study showed that the IR was incapable of mediating cellular transformation when expressed in cells derived from IGFIR-deficient mouse embryos, whereas the IGFIR was capable of mediating this response (9). Studies on signal divergence have largely focused on the COOH-terminal region of the receptors, since this region is the most disparate (10 -15). These studies concluded that the COOH terminus of the IR was important for regulation of a variety of cellular effects but few conclusions regarding the role of the COOH terminus of the IGFIR were proposed. Howev...

DNA cloned from the D. melanogaster (Oregon R) heat shock loci at 63BC and 95D codes for the 83,000 and the 68,000 dalton heat shock proteins, respectively. Both coding sequences occur once per haploid genome. Sequences complementary to messenger RNA for the 70,000 dalton heat shock protein are represented five times, twice at 87A and three times at 87 C. The copies at 87A differ characteristically from those at 87C in an interval of a few hundred bp near the 5' end of the messenger sequence, and the corresponding two classes of hsp 70 messenger RNA are found on polysomes after heat shock. Within this differential region, there is about 15% divergence between messenger sequences cloned from the two loci, while in the rest of the messenger region examined the homology is much closer although still imperfect. Unexpectedly, considerable homology is found between the sequence for the 68,000 dalton heat shock protein at 95D and the sequences for the 70,000 dalton protein at 87A and 87C, and between these sequences and a site in 87D. Messenger RNA molecules of 2.4, 2.55 and 3.05 kb code for the 68,000, 70,000 and 83,000 dalton heat shock proteins and hybridize to apparently uninterrupted DNA sequences of 2.1, 2.25 and 2.6 kb, respectively.

The role of apoptosis in affinity maturation was investigated by determining the affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific antibody-forming cells (AFCs) and serum antibody in transgenic mice that overexpress a suppressor of apoptosis, Bcl-xL, in the B cell compartment. Although transgenic animals briefly expressed higher numbers of splenic AFCs after immunization, the bcl-x L transgene did not increase the number or size of germinal centers (GCs), alter the levels of serum antibody, or change the frequency of NP-specific, long-lived AFCs. Nonetheless, the bcl-x L transgene product, in addition to endogenous Bcl-xL, reduced apoptosis in GC B cells and resulted in the expansion of B lymphocytes bearing VDJ rearrangements that are usually rare in primary anti-NP responses. Long-lived AFCs bearing these noncanonical rearrangements were frequent in the bone marrow and secreted immunoglobulin G1 antibodies with low affinity for NP. The abundance of noncanonical cells lowered the average affinity of long-lived AFCs and serum antibody, demonstrating that Bcl-xL and apoptosis influence clonal selection/maintenance for affinity maturation.

Unique coding sequences for four heat shock proteins of Drosophila melanogaster, hisp 28, hsp 26, hsp 23, and hsp 22, are clustered in a 12-kilobase interval at chromosome subdivision 67B. The four genes are not transcribed in the same direction and each gives rise to a separate messenger RNA, with no indication of intervening sequences. Including the present results, the genes for all seven major heat shock proteins of D. melanogaster are now cloned and are found to exhibit a variety of patterns of organization at the five loci they occupy. The pellet was esended in 10 mM TrisHCI, pH 7.4/10 mM NaCl/10 mM MgCl2/1 mM CaCl2, digested with RNase-free DNase (9), extracted with phenol, precipitated with ethanol, dissolved in H20, mixed with 3 vol of 4 M sodium acetate, pH 6.0, kept on ice 2 hr, and centrifuged 30 min at 4VC at 16,000 X g. The precipitated RNA was dissolved in H20, precipitated with ethanol, and kept at -700C.Plasmid restriction fragments were labeled at the 3' end by using nucleoside [a-32P]triphosphates and 10 units/ml of Escherichia coli DNA polymerase I Klenow fragment (2). Flush-ended restriction fragments were incubated with exonuclease III (gift of W. McClure) for 20 min at 50C (10) before 3'-labeling. Restriction fragments were 5'-labeled by using [a-32P]ATP and polynucleotide kinase (11). 32P-Labeled cDNA was copied from RNA according to Efstratiadis et al. (12 5390The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.