HER-2/c-erbB2 is phosphorylated by calmodulin-dependent protein kinase II on a single site in the cytoplasmic tail at threonine-1172

Calmodulin-dependent protein kinase II (Cam kinase II) is known to desensitise epidermal growth factor receptor (HER-1) tyrosine kinase activity by a process involving phosphorylation at serines 1046/47 in the cytoplasmic tail. We have developed an experimental system to investigate phosphorylation of the related HER-2/c-erbB2 proto-oncogene utilising purified Cam kinase II and recombinant glutathione-S-transferase (GST) fusion proteins. The cDNA for rat Cam kinase II-alpha was transfected into human embryonic kidney (HEK) 293 fibroblasts and the expressed protein purified to homogeneity by calmodulin-agarose affinity chromatography. A GST fusion protein comprising residues 1126-1255 of HER-2 was phosphorylated by purified Cam kinase II, in contrast to a GST protein comprising residues 1005-1125. Phosphoamino-acid analysis and site-directed mutagenesis indicated that HER-2 was phosphorylated on a single site at threonine-1172 which resides within a consensus Cam kinase II phosphorylation site (RAKT). HER-2 (threonine-1172-alanine), in the form of a ligand-inducible chimaera HER-1/2, was co-transfected into HEK-293 fibroblasts with a constitutively active form of Cam kinase II, followed by in vivo labelling of these cells with 32 P-orthophosphate. Immunoprecipitation of ligand-activated receptors followed by two-dimensional phosphopeptide mapping indicated that threonine-1172 in HER-2 is a newly identified in vivo site which can be hyper-phosphorylated by constitutively active Cam kinase II. In addition, when over-expressed in HEK-293 fibroblasts, HER-1/2 (threonine-1172-alanine) showed a defect in desensitisation and underwent a more sustained EGF-induced receptor autophosphorylation compared to wild-type HER-1/2.     

A two-component interpretation of the far infrared dielectric loss in liquid methyl chloroform

A possible interpretation of the so-called Poley absorption in polar liquids is that it is due to overlapping contributions from liquid- lattice bands together with a contribution from an inertially “rolled-off” Debye absorption. We propose a test involving the inertially similar but electrically very different molecules methyl chloroform and carbon tetrachloride. This test gives results which offer plausible support for the two-component interpretation.

     

Macrophage-derived chemokine is a functional ligand for the CC chemokine receptor 4

Macrophage-derived chemokine (MDC) is a recently identified member of the CC chemokine family. MDC is not closely related to other chemokines, sharing most similarity with thymus- and activation-regulated chemokine (TARC), which contains 37% identical amino acids. Both chemokines are highly expressed in the thymus, with little expression seen in other tissues. In addition, the genes for MDC and TARC are encoded by human chromosome 16. To explore this relationship in greater detail, we have more precisely localized the MDC gene to chromosome 16q13, the same position reported for the TARC gene. We have also examined the interaction of MDC with CC chemokine receptor 4 (CCR4), recently shown to be a receptor for TARC. Using a fusion protein of MDC with secreted alkaline phosphatase, we observed high affinity binding of MDC-secreted alkaline phosphatase to CCR4-transfected L1.2 cells (Kd = 0.18 nM). MDC and TARC competed for binding to CCR4, while no binding competition was observed for six other chemokines (MCP-1, MCP-3, MCP-4, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta). MDC was tested for calcium mobilization in L1.2 cells tranfected with seven different CC chemokine receptors. MDC induced a calcium flux in CCR4-transfected cells, but other receptors did not respond to MDC. TARC, which also induced calcium mobilization in CCR4 transfectants, was unable to desensitize the response to MDC. In contrast, MDC fully desensitized a subsequent response to TARC. Both MDC and TARC functioned as chemoattractants for CCR4 transfectants, confirming that MDC is also a functional ligand for CCR4. Since MDC and TARC are both expressed in the thymus, one role for these chemokines may be to attract CCR4-bearing thymocytes in the process of T cell education and differentiation.     

The far infra-red spectrum of crystalline polytetrafluoroethylene

The far infra-red spectrum of highly crystalline polytetrafluoroethylene has been shown to contain a new band near 33cm^?1, the band at 55cm^?1 has been shown to be a close doublet and two further bands at 291 and 308cm^?1 have been observed. These results strongly support the conclusions of Boerio and Koenig from Raman observations that the unit cell contains at least two molecular segments.     

A far infra-red study of conformational disorder in PTFE

The examination of the 250–350 cm^?1 region of the infra-red spectrum of polytetrafluoroethylene (PTFE) has been extended by studies of thermally degraded and sintered material. We conclude that a band at 277 cm^?1 does not arise from a true mode of the crystal but has its origin in regions where the chain has a bent conformation. Our previous speculation that bands at 292 cm^?1 and 312¹ cm^?1 (Raman active only, for the isolated chain) acquire their weak activity in the infra-red through conformational disorder is confirmed. It is likely that the conformational disorder in question arises from chain folding in the polymer crystal.     

On the vibrational assignment problem for polytetrafluoroethylene: 1. The far infra-red spectrum

Studies of the polarized far infra-red spectra of drawn specimens of polytetrafluoroethylene (PTFE) and of the spectrum of the oligomer C₁₄F₃₀ both at room and liquid nitrogen temperatures have given results which strongly confirm the general form of recently published dispersion diagrams. However the absolute values of band intensities and their variation with specimen morphology suggest that some minor changes to the optical intersections are necessary. Intensity arguments also strongly indicate that the labelling, adopted by most recent workers, for the branches B6 and B7 should be interchanged.     

Polarized far infra-red studies of hot-drawn polytetrafluoroethylene

The three strong sharp lines in the 50 cm^?1 region of the infra-red spectrum of polytetrafluoroethylene (PTFE) at liquid nitrogen temperature, have been shown to be strongly polarized, with the transition moments lying perpendicular to the molecular axes. This result is interpreted in terms of the lattice-mode theory and in terms of possible symmetries for the unit cell.     

Far infrared studies of the formation of potassium fluoride dihydrate in and its interaction with low molecular weight polytetrafluorethylene

The process of preparing low molecular weight polytetrafluorethylene by high temperature oxidation with potassium nitrate has been shown to lead to the formation of potassium fluoride in the polymer. This is found to absorb water vapour from the atmosphere forming its dihydrate KF.2H₂O, a crystalline substance which shows a complex many line spectrum in the far infrared. The observed spectrum of KF.2H₂O in the matrix differs in one major respect (the absence of a band at 73 cm^?1) from that of pure KF.2H₂O showing that there is some specific interaction between the dihydrate and its polymeric host.     

Reliable measurement of the optical and dielectric parameters of liquid carbon tetrachloride in the wavelength region

The power absorption coefficient, the refractive index and the loss tangent data for carbon tetrachloride are presented in the region 3 mm–50 μm (3 cm^?1–200 cm^?1). The data are compared with microwave and submillimeter wave literature values. The integrated band intensity and the integrated absorption intensity of the millimeter and submillimeter wave band and octopole moment are calculated and compared.