Ceramide-induced inhibition of T lymphocyte voltage-gated potassium channel is mediated by tyrosine kinases

The n-type K+ channel (n-K+, Kv1.3) in lymphocytes has been recently implicated in the regulation of Fas-induced programmed cell death. Here, we demonstrate that ceramide, a lipid metabolite synthesized upon Fas receptor ligation, inhibits n-K+ channel activity and induces a tyrosine phosphorylation of the Kv1.3 protein in Jurkat T lymphocytes. Tyrosine phosphorylation of the n-K+ channel correlated with an activation of the Src-like tyrosine kinase p56lck upon cellular treatment with the ceramide analog C6-ceramide. Because genetic deficiency of p56lck or inhibition of Src-like tyrosine kinases by herbimycin A prevented ceramide-mediated n-K+ channel inhibition and tyrosine phosphorylation, we propose a ceramide-initiated activation of p56lck resulting in tyrosine phosphorylation and inhibition of the n-K+ channel protein.

The carboxyl terminus of the bacteriophage T4 DNA polymerase is required for holoenzyme complex formation

To further elucidate the mechanism and dynamics of bacteriophage T4 holoenzyme formation, a mutant polymerase in which the last six carboxyl-terminal amino acids are deleted, was constructed, overexpressed, and purified to homogeneity. The mutant polymerase, designated ΔC6 exo−, is identical to wild-type exo− polymerase with respect to kcat, kpol, and dissociation constants for nucleotide and DNA substrate. However, unlike wild-type exo− polymerase, the ΔC6 exo− polymerase is unable to interact with the 45 protein to form the stable holoenzyme. A synthetic polypeptide corresponding to the carboxyl terminus of the wild-type exo− polymerase was tested as an in vitro inhibitor of bacteriophage T4 DNA replication. Surprisingly, the peptide does not directly inhibit holoenzyme complex formation by disrupting the interaction of the polymerase with the 45 protein. On the contrary, the peptide appears to disrupt the interaction of the 44/62 protein with the 45 protein, suggesting that the 44/62 protein and the polymerase use the same site on the 45 protein for functional interactions. Data presented are discussed in terms of a model correlating the functionality of the carboxyl terminus of the polymerase for productive interactions with the 45 protein as well as in terms of the 45 protein concomitantly interacting with the 44/62 protein and polymerase.