Nikos Panayotatos

ERKs: A family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF

We recently described the purification and cloning of extracellular signal-regulated kinase 1 (ERK1), which appears to play a pivotal role in converting tyrosine phosphorylation into the serine/threonine phosphorylations that regulate downstream events. We now describe cloning and characterization of two ERK1-related kinases, ERK2 and ERK3, and provide evidence suggesting that there are additional ERK family members. At least two of the ERKs are activated in response to growth factors; their activations correlate with tyrosine phophorylation, but also depend on additional modifications. Transcripts corresponding to the three cloned ERKs are distinctly regulated both in vivo and in a differentiating cell line. Thus, this family of kinases may serve as intermediates that depend on tyrosine phosphorylation to activate serine/threonine phosphorylation cascades. Individual family members may mediate responses in different developmental stages, in different cell types, or following exposure to different extracellular signals.

Recombinant Human and Rat Ciliary Neurotrophic Factors

The human ciliary neurotrophic factor (CNTF) gene was identified and cloned, based on homology with the recently cloned rat cDNA. The gene encodes a protein of 200 amino acids, which shares about 80% sequence identity with rat and rabbit CNTF and, like these homologues, lacks an apparent secretion signal sequence. The human CNTF gene, like the rat gene, appears to contain a single intron separating two protein coding exons. An intronless human CNTF gene was constructed by the use of polymerase chain reactions and introduced into vectors designed for expression of foreign proteins in E. coli. The rat CNTF gene was also introduced into similar vectors. Both the human and rat proteins were expressed at exceptionally high levels, at 20–40% and 60–70% of total protein, respectively. Extraction of the recombinant proteins from inclusion bodies by guanidinium chloride, followed by two column chromatography steps, produced high yields of pure CNTF that supported survival and neurite outgrowth from embryonic chick ciliary neurons in culture. The biological activity of both recombinant proteins was comparable to that of native rat CNTF.

A Shared, Non-canonical DNA Conformation Detected at DNA/Protein Contact Sites and Bent DNA in the Absence of Supercoiling or Cognate Protein Binding

A hybrid protein (H144), consisting of Lac repressor and T7 endonuclease I, binds at the lac operator and cleaves relaxed double-stranded DNA at distal but distinct sites. These sites are shown here to coincide with a bacterial promoter, a phage T7 promoter, a site for gyrase and intrinsically bent DNA. The targets do not seem to share a particular DNA sequence, and in bent DNA, cleavage occurs at the physical center rather than at the common A-tracts. These results indicate that protein contact sites and intrinsic bends assume a non-canonical conformation in the absence of supercoiling or cognate protein binding. This feature may serve as a recognition signal or facilitate protein binding to initiate transcription and recombination.