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Two cDNAs encoding PAK kinases were isolated from a mouse embryo library by screening with a PCR-generated probe derived from the kinase domain of a rat PAK kinase. These cDNAs, designated PAK-1 and PAK-3, encode mouse PAK kinases of 545 and 544 amino acids, respectively. Both proteins possess an N-terminal Cdc42/Rac interacting binding domain (CRIB) and a C-terminal serine/threonine kinase domain. Comparison of the two mouse PAK kinases revealed that the proteins show 87% amino acid identity. Northern analysis of a multiple mouse tissue blot with a PAK-1 probe detected a 3.0kb transcript that was almost exclusively expressed in the brain and spinal cord compared to other tissues such as lung, liver and kidney. A similar pattern of central nervous system tissue expression of PAK-3 transcripts of 3.6 and 8kb was also observed. Analysis of two multilocus genetic crosses localized Pak1 and Pak3 to a position on chromosome 7 and X, respectively. The high level of PAK-1 and PAK-3 kinase expression in the mouse brain and spinal cord suggests a potentially important role for these kinases in the control of the cellular architecture and/or signaling in the central nervous system.

A limitation of current gene therapy efforts aimed at central nervous system disorders concerns distribution of vectors on direct injection into neural tissue. Here we have circumvented this problem by transferring genes to the meninges surrounding the spinal cord, achieving an in vivo gene transfer paradigm for treating chronic pain. The therapeutic vector consisted of a recombinant adenovirus encoding a secreted form of the potent endogenous opioid beta-endorphin. In an inflammation model of persistent pain, administration of the vector into the cerebrospinal fluid (CSF) surrounding the spinal cord transduced meningeal pia mater cells. The resulting increase in beta-endorphin secretion attenuated inflammatory hyperalgesia, yet had no effect on basal nociceptive responses. This demonstration of a gene transfer approach to pain treatment can be generalized to neurodegenerative disorders in which broad spatial distribution of therapeutic effect is critical.

A plasma membrane iron reductase, required for cellular iron acquisition by Saccharomyces cerevisiae, and the human phagocytic NADPH oxidase, implicated in cellular defense, contain low potential plasma membrane b cytochromes that share elements of structure and function. Four critical histidine residues in the FRE1 protein of the iron reductase were identified by site-directed mutagenesis. Individual mutation of each histidine to alanine eliminated the entire heme spectrum without affecting expression of the apoprotein, documenting the specificity of the requirement for the histidine residues. These critical residues are predicted to coordinate a bis-heme structure between transmembrane domains of the FRE1 protein. The histidine residues are conserved in the related gp91(phox) protein of the NADPH oxidase of human granulocytes, predicting the sites of heme coordination in that protein complex. Similarly spaced histidine residues have also been implicated in heme binding by organelle b cytochromes with little overall sequence similarity to the plasma membrane b cytochromes. This bis-heme motif may play a role in transmembrane electron transport by distinct families of polytopic b cytochromes.

Protein prenylation occurs by modification of proteins with one of at least two isoprenoids, the farnesyl group and the geranylgeranyl group. Protein farnesyltransferases have been identified, but no such enzyme has been identified for geranylgeranylation. We report the identification of an activity in crude soluble yeast extracts that catalyzes the transfer of a geranylgeranyl moiety from geranylgeranyl pyrophosphate to proteins having the C-terminal sequence Cys-Ile-Ile-Leu or Cys-Val-Leu-Leu but not to a similar protein ending with Cys-Ile-Ile-Ser. This activity is dependent upon the CDC43/CAL1 gene, which is involved in budding and the control of cell polarity, but does not require the DPR1/RAM1 gene, which is known to be required for the farnesylation of Ras proteins. These results indicate that the protein geranylgeranyltransferase activity is distinct from the protein farnesyltransferase activity and that its specificity depends in part on the extreme C-terminal leucine in the protein to be prenylated.

The heterotrimeric guanine nucleotide-binding regulatory proteins act at the inner surface of the plasma membrane to relay information from cell surface receptors to effectors inside the cell. These G proteins are not integral membrane proteins, yet are membrane associated. The processing and function of the gamma subunit of the yeast G protein involved in mating-pheromone signal transduction was found to be affected by the same mutations that block ras processing. The nature of these mutations implied that the gamma subunit was polyisoprenylated and that this modification was necessary for membrane association and biological activity. A microbial screen was developed for pharmacological agents that inhibit polyisoprenylation and that have potential application in cancer therapy.