Tumour Necrosis Factor ␣ (TNF␣), is an inflammatory cytokine produced by macrophages/monocytes during acute inflammation and is responsible for a diverse range of signalling events within cells, leading to necrosis or apoptosis. The protein is also important for resistance to infection and cancers. TNF␣ exerts many of its effects by binding, as a trimer, to either a 55 kDa cell membrane receptor termed TNFR-1 or a 75 kDa cell membrane receptor termed TNFR-2. Both these receptors belong to the so-called TNF receptor superfamily. The superfamily includes FAS, CD40, CD27, and RANK. The defining trait of these receptors is an extra cellular domain comprised of two to six repeats of cysteine rich motifs. Additionally, a number of structurally related "decoy receptors" exist that act to sequester TNF molecules, thereby rescuing cells from apoptosis. The crystal structures of TNF␣, TNF␤, the extracellular domain of TNFR-1 (denoted sTNFR-1), and the TNF␤ sTNFR-1 complex have been defined by crystallography. This article will review the structure/function relationships of the TNF␣ and the TNF receptor superfamily. It will also discuss insights as to how structural features play a role in the pleiotropic effects of TNF␣.

P-glycoprotein (Pgp) is a transmembrane transporter causing efflux of a number of chemically unrelated drugs and is responsible for resistance to a variety of anticancer drugs during chemotherapy. Pgp overexpression in cells is also associated with volume-activated chloride channel activity; Pgp is thought to regulate such activity. Reversible phosphorylation is a possible mechanism for regulating the transport and chloride channel regulation functions of Pgp. Protein kinase C (PKC) is a good candidate for inducing such phosphorylation. Hierarchical multiple phosphorylation (e.g. of different serines and with different PKC isoforms) may shuttle the protein between its different states of activity (transport or channel regulation). Cell volume changes may trigger phosphorylation of Pgp at sites causing inhibition of transport. The possible regulation of chloride channels by Pgp and the potential involvement of reversible phosphorylation in such regulation is reviewed.

Phosphorylation in vivo of several proteins in the mammalian heterogeneous nuclear ribonucleoprotein complex (hnRNP), including A1, has been observed and proposed as a regulatory step in pre-mRNA splicing [Maryland, S. H., Dwen, P., & Pederson, T. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7764-7768]. We examined the ability of recombinant hnRNP protein A1 to act as a substrate for a number of purified Ser/Thr protein kinases in vitro. A survey of seven protein kinases showed that A1 was heavily phosphorylated by protein kinase C (PKC) and also was phosphorylated by casein kinase II, protamine kinase, and protein kinase A. In contrast, autophosphorylation-activated protein kinase and two forms of myelin basic protein kinase failed to phosphorylate A1. Proteolysis with trypsin and V8 protease revealed that PKC phosphorylates A1 at three main sites, two in the N-terminal domain (spanning residues 2-196) and one in the C-terminal domain (spanning residues 197-320). Amino acid sequencing revealed that these sites were Ser95, Ser192, and Ser199; phosphorylation at Ser192 was more abundant than at Ser95 and Ser199. Phosphorylation by PKC inhibited the strand annealing activity of A1. Protein phosphatase 2A, but not protein phosphatase 1, dephosphorylated A1 and reversed the inhibitory effect of PKC phosphorylation on the strand annealing activity. A conformational change in the C-terminal domain of A1 was observed upon PKC phosphorylation, and this was associated with a decrease in A1's affinity for single-stranded polynucleotides. The results are consistent with a role of phosphorylation of A1 in regulating its strand annealing activity in vivo.

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