Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase.

A growth factor-stimulated (MAP2-related) protein kinase, ERT, that phosphorylates the epidermal growth factor receptor at Thr669 has been purified from KB human tumor cells by Northwood and co-workers (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). The ERT protein kinase has a restricted substrate specificity, and the structural determinants employed for substrate recognition by this enzyme have not been defined. As an approach toward understanding the specificity of substrate phosphorylation, we have used an in vitro assay to identify additional substrates for the ERT protein kinase. In this report we describe two novel substrates: (a) the human c-myc protein at Ser62 and (b) the rat c-jun protein at Ser246. Alignment of the primary sequences surrounding the phosphorylation sites located within the epidermal growth factor receptor (Thr669), Myc (Ser62), and Jun (Ser246) demonstrated a marked similarity. The observed consensus sequence was Pro-Leu-Ser/Thr-Pro. We propose that this sequence forms part of a substrate structure that is recognized by the ERT protein kinase.     

Mutational analysis of the cytoplasmic tail of the human transferrin receptor. Identification of a sub-domain that is required for rapid endocytosis

It has been reported that the sequence Tyr20-X-Arg-Phe23 present within the cytoplasmic tail of the transferrin receptor may represent a tyrosine internalization signal (Collawn, J.F., Stangel, M., Kuhn, L.A., Esekogwu, V., Jing, S., Trowbridge, I.S., and Tainer, J. A. (1990) Cell 63, 1061-1072). However, as Tyr20 is not conserved between species (Alvarez, E., Gironès, N., and Davis, R. J. (1990) Biochem. J. 267, 31-35), the functional role of the putative tyrosine internalization signal is not clear. To address this question, we constructed a series of 32 deletions and point mutations within the cytoplasmic tail of the human transferrin receptor. The effect of these mutations on the apparent first order rate constant for receptor endocytosis was examined. It was found that the region of the cytoplasmic tail that is proximal to the transmembrane domain (residues 28-58) is dispensable for rapid endocytosis. In contrast, the distal region of the cytoplasmic tail (residues 1-27) was found to be both necessary and sufficient for the rapid internalization of the transferrin receptor. The region identified includes Tyr20-X-Arg-Phe23, but is significantly larger than this tetrapeptide. It is therefore likely that structural information in addition to the proposed tyrosine internalization signal is required for endocytosis. To test this hypothesis, we investigated whether a heterologous tyrosine internalization signal (from the low density lipoprotein receptor) could function to cause the rapid endocytosis of the transferrin receptor. It was observed that this heterologous tyrosine internalization signal did not allow rapid endocytosis. We conclude that the putative tyrosine internalization signal (Tyr20-Thr-Arg-Phe23) is not sufficient to determine rapid endocytosis of the transferrin receptor. The data reported here indicate that the transferrin receptor internalization signal is formed by a larger cytoplasmic tail structure located at the amino terminus of the receptor.     

ICAM-2 peptides mediate lymphocyte adhesion by binding to CD11a/CD18 and CD49d/CD29 integrins.

Three fifteen-amino-acid polypeptides designated peptides 1, 2 and 3 were synthesised as likely candidates for mimicking the role of ICAM-2 as a ligand. The ability of each peptide to bind lymphoid cells was tested. Peptide 2 largely mediated cell attachment of unstimulated cells and this binding was only marginally increased by stimulating the cells with phorbol dibutyrate (P(Bu)2). Peptide 3 mediated minimal spontaneous cell attachment, but this binding was significantly enhanced following P(Bu)2 stimulation. Peptide 1 had no effect on cell attachment with or without stimulation. The cell attachment to peptide 2 was both temperature- and cation-dependent. Studies using specific monoclonal antibodies showed that with unstimulated cells, anti-VLA-4 alpha(CD49d) or beta chain (CD29) antibodies (KD4-13 and 4B4) and anti-CD18 (1B4) each partially inhibited the cell binding. Monoclonal antibodies against CD54 (ICAM-1; 84H10 or LB2), MHC class 1 (W6/32) and control mouse IgG had no effect. When anti-CD29 and anti-CD18 monoclonal antibodies were used concurrently, there was almost complete inhibition of the cell attachment. These observations indicated that cell adhesion via ICAM-2 is mediated: (i) predominantly by peptide 2 in unstimulated and P(Bu)2-stimulated cells, and also, to some extent, by peptide 3 in P(Bu)2-stimulated cells and (ii) by binding to both CD11/CD18 and CD49d/CD29 integrins.     

Isoproterenol fails to produce myocardial necrosis in streptozotocin-induced diabetic rats.

Biochemical alterations in the hearts of non-diabetic and 5 weeks of streptozotocin-induced diabetic rats following isoproterenol (ISO) administration were compared. Serum lactate dehydrogenase (LDH) and myocardial adenosine triphosphate (ATP), creatine phosphate (CP), lactate and glycogen were used as indices of myocardial injury. Hearts from diabetic rats (blood glucose greater than 350 mg/dl), before ISO administration, had normal lactate levels but significantly low high-energy phosphate (HEP) levels and high glycogen levels in comparison to non-diabetic rats. No difference was observed in serum LDH levels between these two groups. ISO administration to non-diabetic rats caused myocardial necrosis as evidenced by a significant depletion of myocardial glycogen and HEPs along with significant myocardial lactate accumulation and an increase in serum LDH. However, the hearts from diabetic rats failed to show any significant HEP depletion, accumulation of lactate and leakage of LDH into serum following ISO-administration, though myocardial glycogen level was significantly lowered. These observations, in conjunction with earlier reports, point to the hypothesis that, in diabetes, there are certain alterations in the sarcolemma which hamper the process by which ISO causes myocardial necrosis.     

Amino acid deprivation induces synthesis of four proteins in chick embryo cells

Amino acid deprivation of chick embryo cells enhances the synthesis of four proteins whose molecular weights are approx. 89000, 73000, 35000 and 27000. This enhancement, which is seen in medium completely free of amino acids, can be prevented by the addition of any single amino acid. Furthermore, in the absence of amino acids in the medium, DNA and RNA synthesis is markedly inhibited, an effect which is similarly prevented by the addition of single amino acids. These new proteins synthesized in the amino acid-free medium co-migrate on one-dimensional gels with the ‘stress proteins’ induced by a variety of agents such as heavy metals, sulfhydryl reagents, heat shock, and amino acid analogues.     

A stochastic compartmental model with continuous infusion

This paper deals with stochasticm-compartmental systems with continuous time-dependent infusions into all compartments and reversible time-independent flows between any two compartments. A methodology for the first two moments of the distribution of the number of units in the different compartments at any point in time is outlined without resorting to the usual techniques of generating functions and inverse Laplace transforms. A possible application to a systems analysis of the kidney transplant system is discussed.     

Group-specific component in Macaca.

The group-specific component in 17 Macaca mulatta was examined. All the individuals revealed the same Gc 1--1 pattern.     

The impacts of cytoplasmic incompatibility factor (cifA and cifB) genetic variation on phenotypes

Wolbachia are maternally transmitted, intracellular bacteria that can often selfishly spread through arthropod populations via cytoplasmic incompatibility (CI). CI manifests as embryonic death when males expressing prophage WO genes cifA and cifB mate with uninfected females or females harboring an incompatible Wolbachia strain. Females with a compatible cifA-expressing strain rescue CI. Thus, cif-mediated CI confers a relative fitness advantage to females transmitting Wolbachia. However, whether cif sequence variation underpins incompatibilities between Wolbachia strains and variation in CI penetrance remains unknown. Here, we engineer Drosophila melanogaster to transgenically express cognate and non-cognate cif homologs and assess their CI and rescue capability. Cognate expression revealed that cifA;B native to D. melanogaster causes strong CI, and cognate cifA;B homologs from two other Drosophila-associated Wolbachia cause weak transgenic CI, including the first demonstration of phylogenetic type 2 cifA;B CI. Intriguingly, non-cognate expression of cifA and cifB alleles from different strains revealed that cifA homologs generally contribute to strong transgenic CI and interchangeable rescue despite their evolutionary divergence, and cifB genetic divergence contributes to weak or no transgenic CI. Finally, we find that a type 1 cifA can rescue CI caused by a genetically divergent type 2 cifA;B in a manner consistent with unidirectional incompatibility. By genetically dissecting individual CI functions for type 1 and 2 cifA and cifB, this work illuminates new relationships between cif genotype and CI phenotype. We discuss the relevance of these findings to CI’s genetic basis, phenotypic variation patterns, and mechanism.