The serine/threonine kinase RAF‐1 is phosphorylated in intact macrophages in response to CSF‐1 at 37 degrees C The augmented phosphorylation of RAF‐1 and a concomitant increase in RAF‐1 associated serine/threonine kinase activity are kinetically later events than CSF‐1 induced protein tyrosine phosphorylation. Furthermore, phosphoamino acid analysis of RAF‐1 reveals the presence of phosphoserine, trace amounts of phosphothreonine but no phosphotyrosine and the phosphorylated RAF‐1 does not react with anti‐phosphotyrosine antibodies. In contrast to CSF‐1 induced protein tyrosine phosphorylation, RAF‐1 phosphorylation and activation are temperature dependent and do not occur at 4 degrees C. Furthermore, coprecipitation experiments failed to reveal any noncovalent association of RAF‐1 with the CSF‐1 receptor. Thus, while RAF‐1 is not a direct substrate for the CSF‐1 receptor tyrosine kinase in vivo, its temperature dependent phosphorylation and activation represent an intriguing aspect of the CSF‐1 response.

In this paper we have studied a PDZ protein domain as a possible tool for cellular targeting of the ribosome inactivating protein Saporin, exploiting the ability of PDZ domains to recognize and bind short peptide sequences located at the C-terminus of a cognate protein. We have focused our attention on the PDZ domain from hCASK (Human calcium/calmodulin-dependent serine protein kinase) that binds extracellular CD98 in epithelial cells, being this antigen recognized as a marker for several human tumors and particularly considered a negative prognostic marker for human glioblastoma. We produced recombinant fusions of one or two hCASK-PDZ domains with the ribosome inactivating protein Saporin and assayed them on two human glioblastoma cell lines (GL15 and U87). These constructs proved to be toxic, with increasing activity as a function of the number of PDZ domains, and induce cell death by apoptotic mechanisms in a dose-dependent and/or time dependent manner.

No abstract

No abstract