Human calcium-calmodulin dependent protein kinase I: cDNA cloning, domain structure and activation by phosphorylation at threonine-177 by calcium-calmodulin dependent protein kinase I kinase.

Human Ca(2+)-calmodulin (CaM) dependent protein kinase I (CaMKI) encodes a 370 amino acid protein with a calculated M(r) of 41,337. The 1.5 kb CaMKI mRNA is expressed in many different human tissues and is the product of a single gene located on human chromosome 3. CaMKI 1-306, was unable to bind Ca(2+)-CaM and was completely inactive thereby defining an essential component of the CaM-binding domain to residues C-terminal to 306. CaMKI 1-294 did not bind CaM but was fully active in the absence of Ca(2+)-CaM, indicating that residues 295-306 are sufficient to maintain CaMKI in an auto-inhibited state. CaMKI was phosphorylated on Thr177 and its activity enhanced approximately 25-fold by CaMKI kinase in a Ca(2+)-CaM dependent manner. Replacement of Thr177 with Ala or Asp prevented both phosphorylation and activation by CaMKI kinase and the latter replacement also led to partial activation in the absence of CaMKI kinase. Whereas CaMKI 1-306 was unresponsive to CaMKI kinase, the 1-294 mutant was phosphorylated and activated by CaMKI kinase in both the presence and absence of Ca(2+)-CaM although at a faster rate in its presence. These results indicate that the auto-inhibitory domain in CaMKI gates, in a Ca(2+)-CaM dependent fashion, accessibility of both substrates to the substrate binding cleft and CaMKI kinase to Thr177. Additionally, CaMKI kinase responds directly to Ca(2+)-CaM with increased activity.     

Chemoattractant receptors activate distinct pathways for chemotaxis and secretion. Role of G-protein usage

Human leukocyte chemoattractant receptors activate chemotactic and cytotoxic pathways to varying degrees and also activate different G-proteins depending on the receptor and the cell-type. To determine the relationship between G-protein usage and the biological and biochemical responses activated, receptors for the chemoattractants formyl peptides (FR), platelet-activating factor (PAFR), and leukotriene B(4) (BLTR) were transfected into RBL-2H3 cells. Pertussis toxin (Ptx) served as a Galpha(i) inhibitor. These receptors were chosen to represent the spectrum of G(i) usage as Ptx had differential effects on their ability to induce calcium mobilization, phosphoinositide hydrolysis, and exocytosis with complete inhibition of all responses by FR, intermediate effects on BLTR, and little effect on PAFR. Ptx did not affect ligand-induced phosphorylation of PAFR and BLTR but inhibited phosphorylation of FR. In contrast, chemotaxis to formylmethionylleucylphenylalanine, leukotriene B(4), and platelet-activating factor was completely blocked by Ptx. Wortmannin, a phosphotidylinositol 3-kinase inhibitor, also completely blocked ligand-induced chemotaxis by all receptors but did not affect calcium mobilization or phosphoinositide hydrolysis; however, it partially blocked the exocytosis response to formylmethionylleucylphenylalanine and the platelet-activating factor. Membrane ruffling and pseudopod extension via the BLTR was also completely inhibited by both Ptx and wortmannin. These data suggest that of the chemoattractant receptors studied, G-protein usage varies with FR being totally dependent on G(i), whereas BLTR and PAFR utilize both G(i) and a Ptx-insensitive G-protein. Both Ptx-sensitive and -insensitive G-protein usage can mediate the activation of phospholipase C, mobilization of intracellular calcium, and exocytosis by chemoattractant receptors. Chemotaxis, however, had an absolute requirement for a G(i)-mediated pathway.     

Cognitive empathy in inter-disciplinary research: The contrasting attitudes of plant breeders and molecular biologists towards rice

I draw attention to the perceptions of and interactions between molecular biologists and scientists engaged in plant breeding in India, who have been attempting to employ molecular biology tools to understand and intervene to improve the rice crop. The present essay suggests that the concept of cognitive empathy is crucial for enabling basic scientists and applied scientists to begin to understand phenomena from the point of view of the other and from the point of view of society at large, and in arriving at solutions that are scientifically feasible and socially acceptable. Socialization into disciplinary cultures, organizational factors and individual anxieties seem to inhibit inter-disciplinary collaboration. The majority of rice breeders and a small group of molecular biologists emphasize the relative merits of marker-assisted selection (MAS) in the near term vis-à-vis the currently controversial transgenic approach for rice crop improvement in India. An earlier version was presented at the International Transdisciplinarity Conference held at the Swiss Federal Institute of Technology, Zürich, Switzerland, February 27–March 1, 2000.     

Flexibility and curvature of duplex DNA containing mismatched sites as a function of temperature

The flexibility and curvature of duplex DNAs containing mismatched sites have been monitored as a function of temperature. The diffusion coefficients are dependent on the flexibility and the curvature of the DNA, and these have been determined by NMR-based methods. The diffusion coefficients, D, depend on a Boltzmann term and the viscosity of the solvent, eta, which is also temperature dependent. To analyze the temperature dependence of the diffusion results, the shape function, S(f) = etaD/T, is used. The shape functions do not have the viscosity and temperature dependence of the diffusion coefficients. The presence of mismatched sites significantly enhances the shape function of duplex DNA at all temperatures examined. The observed increases in the shape functions are attributed to the mismatched sites acting as localized flexible joints. The results on the temperature dependence of the shape functions, the optical absorbance, and the proton chemical shifts indicate that local melting at, and adjacent to, mismatched site occurs at a lower temperature than the overall melting of the duplexes. The localized melting gives rise to a considerable increase in the shape function. The contribution of the curvature of the mismatched sites to the enhanced diffusion has been examined. A DNA with mismatches that are in phase with respect to the helical repeat and a DNA which has the mismatches out of phase with respect to the helical repeat have been examined. The results indicate that mismatched sites have modest curvature.     

beta-Arrestin-dependent constitutive internalization of the human chemokine decoy receptor D6

Seven transmembrane receptors mediate diverse physiological responses including hormone action, olfaction, neurotransmission, and chemotaxis. Human D6 is a non-signaling seven-transmembrane receptor expressed on lymphatic endothelium interacting with most inflammatory CC-chemokines resulting in their rapid internalization. Here, we demonstrate that this scavenging activity is mediated by continuous internalization and constant surface expression of the receptor, a process involving the clathrin-coated pit-dependent pathway. D6 constitutively associates with the cytoplasmic adaptor beta-arrestin, and this interaction is essential for D6 internalization. An acidic region, but not the putative phosphorylation sites in the cytoplasmic tail of D6, is critical for receptor interaction with beta-arrestin and subsequent internalization. Neither the native D6 nor mutants uncoupled from beta-arrestin activate any G-protein-mediated signaling pathways. Therefore, D6 may be considered a decoy receptor structurally adapted to perform chemokine scavenging.