A sphingosine-dependent protein kinase that specifically phosphorylates 14-3-3 (SDK1) is identified as the kinase domain of PKCdelta: a preliminary note

A specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, eta, or zeta, respectively, only in the presence of sphingosine (Sph) or N,N-dimethyl-Sph (DMS), was termed "sphingosine-dependent protein kinase-1" (SDK1) [J. Biol. Chem. 273(34) (1998) 21834]. We have now identified SDK1 as a protein having the same amino acid sequence as in the C-terminal-half kinase domain of PKCdelta, with approximately 40 kDa molecular mass, based on large-scale purification of a protein from rat liver, and partial sequence using three different combinations of LC-MS or LC-MS/MS with respective search engine. PKCdelta did not display any SDK1 activity and PKCdelta activity was inhibited by Sph and DMS. However, strong SDK1 activity, only in the presence of Sph or DMS, became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40 kDa kinase domain.     

Characterization of the mouse gene for the U-box-type ubiquitin ligase UFD2a

UFD2a is a mammalian homolog of Saccharomyces cerevisiae Ufd2, originally described as an E4 ubiquitination factor. UFD2a belongs to the U-box family of ubiquitin ligases (E3s) and likely functions as both an E3 and E4. We have isolated and characterized the mouse gene (Ube4b) for UFD2a. A full-length (approximately 5700 bp) Ube4b cDNA was isolated and the corresponding gene spans >100 kb, comprising 27 exons. Luciferase reporter gene analysis of the 5(') flanking region of Ube4b revealed that nucleotides -1018 to -943 (relative to the translation initiation site) possess promoter activity. This functional sequence contains two putative Sp1 binding sites but not a TATA box. Immunoblot and immunohistochemical analyses revealed that UFD2a is expressed predominantly in the neuronal tissues. We also show that UFD2a interacts with VCP (a AAA-family ATPase) that is thought to mediate protein folding. These data implicate UFD2a in the degradation of neuronal proteins by the ubiquitin-proteasome pathway.     

A role of the Duffy antigen for the maintenance of plasma chemokine concentrations

We examined plasma chemokine concentrations and chemokine clearance rates in Duffy antigen knockout mice. The plasma concentrations of eotaxin and MCP-1 in Duffy antigen knockout mice were less than one-third of those in wild-type mice. When eotaxin or hMGSA was intravenously injected, the chemokine disappeared more rapidly from the plasma of Duffy antigen knockout mice than from the plasma of wild-type mice. The half-lives of hIP-10 and interferon-gamma, which do not have an affinity for the Duffy antigen, in plasma were indistinguishable between Duffy antigen knockout mice and wild-type mice. These results suggest that the Duffy antigen delays the disappearance of chemokines from the plasma, resulting in the maintenance of plasma chemokine concentrations.     

Human Fc epsilon RIalpha-specific human single-chain Fv (scFv) antibody with antagonistic activity toward IgE/Fc epsilon RIalpha-binding

The alpha-chain of Fc epsilon RI (Fc epsilon RIalpha) plays a critical role in the binding of IgE to Fc epsilon RI. A fully human antibody interfering with this interaction may be useful for the prevention of IgE-mediated allergic diseases. Here, we describe the successful isolation of a human single-chain Fv antibody specific to human Fc epsilon RIalpha using human antibody phage display libraries. Using the non-immune phage antibody libraries constructed from peripheral blood lymphocyte cDNA from 20 healthy subjects, we isolated three phage clones (designated as FcR epsilon 27, FcR epsilon 51, and FcR epsilon 70) through two rounds of biopanning selection. The purified soluble scFv, FcR epsilon 51, inhibited the binding of IgE to recombinant Fc epsilon RIalpha, although both FcR epsilon 27 and FcR epsilon 70 showed fine binding specificity to Fc epsilon RIalpha. Since FcR epsilon 51 was determined to be a monomer by HPLC, BIAcore analysis was performed. The dissociation constant of FcR epsilon 51 to Fc epsilon RIalpha was estimated to be 20 nM, i.e., fortyfold lower than that of IgE binding to Fc epsilon RIalpha (K(d) = 0.5 nM). With these characteristics, FcR epsilon 51 exhibited inhibitory activity on the release of histamine from passively sensitized human peripheral blood mononuclear cells.     

Ubiquitylation as a quality control system for intracellular proteins

Quality control of intracellular proteins is essential for cellular homeostasis. Molecular chaperones recognize and contribute to the refolding of misfolded or unfolded proteins, whereas the ubiquitin-proteasome system mediates the degradation of such abnormal proteins. Ubiquitin-protein ligases (E3s) determine the substrate specificity for ubiquitylation and have been classified into HECT and RING-finger families. More recently, however, U-box proteins, which contain a domain (the U box) of about 70 amino acids that is conserved from yeast to humans, have been identified as a new type of E3. The prototype U-box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and an E3 to catalyze the formation of a ubiquitin chain on artificial substrates. Yeast Ufd2 is functionally implicated in cell survival under stressful conditions. This review addresses recent progress in characterization of the role of E3 enzymes, especially that of U-box proteins, in quality control of intracellular proteins.     

Chemo-enzymatic synthesis of 3-(2-naphthyl)- L-alanine by an aminotransferase from the extreme thermophile, Thermococcus profundus

Hyper-thermostable aminotransferase from Thermococcus profundus (MsAT) was used to synthesize 3-(2-naphthyl)-l-alanine (Nal) by transamination between its corresponding -keto acid, 3-(2-naphthyl)pyruvate (NPA) and l-glutamate (Glu) at 70 °C. Equilibrium of this reaction was shifted toward Nal production due to its low solubility, giving rise to Nal precipitate. Optically pure Nal (>99% ee) was synthesized with 93% (mol mol^–1) yield from 180 mM NPA and 360 mM Glu.     

Molecular evidence for two vitellogenin genes and processing of vitellogenins in the American cockroach, Periplaneta americana

The American cockroach, Periplaneta americana has two vitellins (Vn1 and Vn2) and corresponding vitellogenins (Vg1 and Vg2). Vns/Vgs were separated on the SDS-PAGE as three major polypeptide bands [170, 100 (multisubunits), and 50 kD] and a minor polypeptide band (150 kD) both in the egg (mature terminal oocyte) extract and in the female hemolymph. We previously cloned one Vg (Vg1) cDNA and showed that the 170-kD polypeptide originated from the C-terminus of the Vg1. In the present study, we cloned the other Vg (Vg2) cDNA. It is 5,826 bp long encoding 1,876 amino acid residues (including 16 residues for putative signal peptide) in a single ORF. The deduced amino acid sequences of both Vgs (Vg1 and Vg2) of P. americana showed 30% identity. The GL/ICG motif is followed by eight cysteine residues at conserved locations near the C-terminal and the DGXR motif starts 18 residues upstream of the GL/ICG motif. The chemically determined N-terminal amino acid sequences of the 150-kD and of the 50-kD polypeptides matched exactly with each other and with the deduced N-terminal amino acid sequence of the Vg2 cDNA. The pattern of processing in P. americana Vns/Vgs is discussed.