No abstract

The p27Kip1 protein associates with G1‐specific cyclin–CDK complexes and inhibits their catalytic activity. p27Kip1 is regulated at various levels, including translation, degradation by the ubiquitin/proteasome pathway and non‐covalent sequestration. Here, we describe point mutants of p27 deficient in their interaction with either cyclins (p27c−), CDKs (p27k−) or both (p27ck−), and demonstrate that each contact is critical for kinase inhibition and induction of G1 arrest. Through its intact cyclin contact, p27k− associated with active cyclin E–CDK2 and, unlike wild type p27, p27c− or p27ck−, was efficiently phosphorylated by CDK2 on a conserved C‐terminal CDK target site (TPKK). Retrovirally expressed p27k− was rapidly degraded through the proteasome in Rat1 cells, but was stabilized by secondary mutation of the TPKK site to VPKK. In this experimental setting, exogenous wild‐type p27 formed inactive ternary complexes with cellular cyclin E–CDK2, was not degraded through the proteasome, and was not further stabilized by the VPKK mutation. p27ck−, which was not recruited to cyclin E–CDK2, also remained stable in vivo. Thus, selective degradation of p27k− depended upon association with active cyclin E–CDK2 and subsequent phosphorylation. Altogether, these data show that p27 must be phosphorylated by CDK2 on the TPKK site in order to be degraded by the proteasome. We propose that cellular p27 must also exist transiently in a cyclin‐bound non‐inhibitory conformation in vivo.

We show here that c‐Myc antagonizes the cyclin‐dependent kinase (CDK) inhibitor p27Kip1. p27 expressed from recombinant retroviruses in Rat1 cells associated with and inhibited cyclin E/CDK2 complexes, induced accumulation of the pRb and p130 proteins in their hypophosphorylated forms, and arrested cells in G1. Prior expression of c‐Myc prevented inactivation of cyclin E/CDK2 as well as dephosphorylation of pRb and p130, and allowed continuous cell proliferation in the presence of p27. This effect did not require ubiquitin‐mediated degradation of p27. Myc altered neither the susceptibility of cyclin E/CDK2 to inhibition by p27, nor the intrinsic CDK‐inhibitory activity of p27, but induced sequestration of p27 in a form unable to bind cyclin E/CDK2. Neither Myc itself nor other G1‐cyclin/CDK complexes were directly responsible for p27 sequestration. Retroviral expression of G1 cyclins (D1–3, E or A) or of the Cdc25A phosphatase did not overcome p27‐induced arrest. Growth rescue by Myc required dimerization with Max, DNA binding and an intact transcriptional activation domain, as previously shown for cellular transformation. We propose that this activity is mediated by the product of an as yet unknown Myc‐Max target gene(s) and represents an essential aspect of Myc's mitogenic and oncogenic functions.

DNA sequence analysis of a 12236 bp fragment, which is located upstream of nifE in Rhodobacter capsulatus nif region A, revealed the presence of ten open reading frames. With the exception of fdxC and fdxN, which encode a plant-type and a bacterial-type ferredoxin, the deduced products of these coding regions exhibited no significant homology to known proteins. Analysis of defined insertion and deletion mutants demonstrated that six of these genes were required for nitrogen fixation. Therefore, we propose to call these genes rnfA, rnfB, rnfC, rnfD, rnfE and rnfF (for Rhodobacter nitrogen fixation). Secondary structure predictions suggested that the rnf genes encode four potential membrane proteins and two putative iron-sulphur proteins, which contain cysteine motifs (C-X2-C-X2-C-X3-C-P) typical for [4Fe--4S] proteins. Comparison of the in vivo and in vitro nitrogenase activities of fdxN and rnf mutants suggested that the products encoded by these genes are involved in electron transport to nitrogenase. In addition, these mutants were shown to contain significantly reduced amounts of nitrogenase. The hypothesis that this new class of nitrogen fixation genes encodes components of an electron transfer system to nitrogenase was corroborated by analysing the effect of metronidazole. Both the fdxN and rnf mutants had higher growth yields in the presence of metronidazole than the wild type, suggesting that these mutants contained lower amounts of reduced ferredoxins.

Retroviral expression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4a) in rodent fibroblasts induces dephosphorylation of pRb, p107 and p130 and leads to G1 arrest. Prior expression of cyclin E allows S-phase entry and long-term proliferation in the presence of p16. Cyclin E prevents neither the dephosphorylation of pRb family proteins, nor their association with E2F proteins in response to p16. Thus, cyclin E can bypass the p16/pRb growth-inhibitory pathway downstream of pRb activation. Retroviruses expressing E2F-1, -2 or -3 also prevent p16-induced growth arrest but are ineffective against the cyclin E-CDK2 inhibitor p27(Kip1), suggesting that E2F cannot substitute for cyclin E activity. Thus, cyclin E possesses an E2F-independent function required to enter S-phase. However, cyclin E may not simply bypass E2F function in the presence of p16, since it restores expression of E2F-regulated genes such as cyclin A or CDC2. Finally, c-Myc bypasses the p16/pRb pathway with effects indistinguishable from those of cyclin E. We suggest that this effect of Myc is mediated by its action upstream of cyclin E-CDK2, and occurs via the neutralization of p27(Kip1) family proteins, rather than induction of Cdc25A. Our data imply that oncogenic activation of c-Myc, and possibly also of cyclin E, mimics loss of the p16/pRb pathway during oncogenesis.

The envelope glycoprotein (Env) complex of HIV-1 undergoes rapid internalization from the plasma membrane of human cells by virtue of a tyrosine-based endocytic signal (RQGYSPL, residues 704-710) in the cytosolic tail of the protein (J. F. Rowell et al., J. Immunol. 155, 473-488, 1995). Here we demonstrate that this tyrosine-based signal interacts with the mu 2 (medium) chain of the AP-2 clathrin-associated adaptor, a protein complex involved in endocytosis of cell surface receptors. The same signal is also capable of interacting with two other members of the adaptor medium chain family, mu 1 and mu 3A, which are components of the AP-1 and AP-3 adaptor complexes, respectively. Interactions with mu 1 and mu 3A might be responsible for the targeting of the internalized envelope glycoprotein to lysosomes or to the basolateral plasma membrane of polarized epithelial cells. A second potential tyrosine-based signal (LFSYHRL, residues 760-766) also interacts with mu 1, mu 2, and mu 3A, although it is less important for internalization in vivo probably due to its position within the cytosolic tail. Overexpression of chimeric proteins having the HIV-1 Env cytosolic tail increases expression of the transferrin receptor on the cell surface, probably due to saturation of the cellular pool of mu 2 by the overexpressed proteins. These observations suggest that HIV-1 Env utilizes the protein sorting machinery of the host cells for internalization and sorting at various steps of the endocytic and biosynthetic pathways.

Two new thermosensitive yeast mutants defective in retrieval of dilysine‐tagged proteins from the Golgi back to the endoplasmic reticulum (ER) were characterized. While both ret2–1 and ret3–1 were defective for ER retrieval, only ret2–1 exhibited a defect in forward ER‐to‐Golgi transport at the non‐permissive temperature. Coatomer (COPI) from both mutants could efficiently bind dilysine motifs in vitro. The corresponding RET2 and RET3 genes were cloned by complementation and found of encode the delta and zeta subunits of coatomer respectively. Both proteins show significant homology to clathrin adaptor subunits. These results emphasize the role of coatomer in retrieval of dilysine‐tagged proteins back to the ER, and the similarity between clathrin and coatomer coats.

Abstract. Signals that can cause retention in the ER have been found in the cytoplasmic domain of individual subunits of multimeric receptors destined to the cell surface. To study how ER retention motifs are masked during assembly of oligomeric receptors, we analyzed the assembly and intracellular transport of the human high-affinity receptor for immunoglobulin E expressed in COS cells. The cytoplasmic domain of the ot chain contains a dilysine ER retention signal, which becomes nonfunctional after assembly with the 3' chain, allowing transport out of the ER of the fully assembled receptor. Juxtaposition of the cytoplasmic domains of the, and ~/subunits during assembly is responsible for this loss of ER retention. Substitution of the 3' chain cytoplasmic domain with cytoplasmic domains of irrelevant proteins resulted in efficient transport out of the ER of the ot chain, demonstrating that nonspecific steric hindrance by the cytoplasmic domain of the 7 chain accounts for the masking of the ER retention signal present in the cytoplasmic domain of the o~ chain. Such a mechanism allows the ER retention machinery to discriminate between assembled and nonassembled receptors, and thus participates in quality control at the level of the ER.