Molecular interactions of the Gbeta binding domain of the Ste20p/PAK family of protein kinases. An isolated but fully functional Gbeta binding domain from Ste20p is only partially folded as shown by heteronuclear NMR spectroscopy.

The transmission of the mating signal of the budding yeast Saccharomyces cerevisiae requires Ste20p, a member of the serine/threonine protein kinases of the Ste20p/PAK family, to link the Gbeta subunit of the heterotrimeric G protein to the mitogen-activated protein kinase cascades. The binding site of Ste20p to the Gbeta subunit was mapped to a consensus sequence of SSLphiPLI/VXphiphibeta (X for any residue; phi for A, I, L, S or T; beta for basic residues), which was shown to be a novel Gbeta binding (GBB) motif present only in the noncatalytic C-terminal domains of the Ste20p/PAK family of protein kinases (Leeuw, T., Wu, C., Schrag, J. D., Whiteway, M., Thomas, D. Y., and Leberer, E. (1998) Nature 391, 191-195; Leberer, E., Dignard, D., Thomas, D. Y., and Leeuw, T. (2000) Biol. Chem. 381, 427-431). Here, we report the results of an NMR study on two GBB motif peptides and the entire C-terminal domain derived from Ste20p. The NMR data show that the two peptide fragments are not uniquely structured in aqueous solution, but in the presence of 40% trifluoroethanol, the longer 37-residue peptide exhibited two well defined, but flexibly linked helical structure elements. Heteronuclear NMR data indicate that the fully functional 86-residue C-terminal domain of Ste20p is again unfolded in aqueous solution but has helical secondary structure preferences similar to those of the two peptide fragments. The NMR results on the two GBB peptides and the entire GBB domain all indicate that the two important binding residues, Ser(879) and Ser(880), are located at the junction between two helical segments. These experimental observations with the prototype GBB domain of a novel family of Gbeta-controlled effectors may have important implications in understanding the molecular mechanisms of the signal transduction from the heterotrimeric G protein to the mitogen-activated protein kinase cascade.

Purification, cDNA cloning, and expression of a new human blood plasma glutamate carboxypeptidase homologous to N-acetyl-aspartyl-alpha-glutamate carboxypeptidase/prostate-specific membrane antigen.

We describe the identification, cDNA cloning, and biochemical characterization of a new human blood plasma glutamate carboxypeptidase (PGCP). PGCP was co-purified from human placenta with lysosomal carboxypeptidase, cathepsin A, lysosomal endopeptidase, cathepsin D, and a gamma-interferon-inducible protein, IP-30, using an affinity chromatography on a Phe-Leu-agarose column. A PGCP cDNA was obtained as an expressed sequence tag clone and completed at 5'-end by rapid amplification of cDNA ends polymerase chain reaction. The cDNA contained a 1623-base pair open reading frame predicting a 541-amino acid protein, with five putative Asn glycosylation sites and a 21-residue signal peptide. PGCP showed significant amino acid sequence homology to several cocatalytic metallopeptidases including a glutamate carboxypeptidase II also known as N-acetyl-aspartyl-alpha-glutamate carboxypeptidase or as prostate-specific membrane antigen and expressed glutamate carboxypeptidase activity. Expression of the PGCP cDNA in COS-1 cells, followed by Western blotting and metabolic labeling showed that PGCP is synthesized as a 62-kDa precursor, which is processed to a 56-kDa mature form containing two Asn-linked oligosaccharide chains. The mature form of PGCP was secreted into the culture medium, which is consistent with its intracellular localization in secretion granules. In humans, PGCP is found principally in blood plasma, suggesting a potential role in the metabolism of secreted peptides.

Novel dimeric Nur77 signaling mechanism in endocrine and lymphoid cells.

Within the nuclear receptor family, Nur77 (also known as NGFI-B) distinguishes itself by its ability to bind a target sequence (the NBRE) as a monomer and by its role in T-cell receptor (TCR)-induced apoptosis in T cells. We now report on a novel mechanism of Nur77 action that is mediated by homodimers. These dimers bind a Nur77 response element (NurRE), which has been identified as a target of CRH-induced Nur77 in the pro-opiomelanocortin (POMC) gene promoter. Both halves of the palindromic NurRE are required for responsiveness to physiological signals, like CRH in pituitary-derived AtT-20 cells. Similarly, in T-cell hybridomas, TCR activation induced NurRE but not NBRE reporters. The in vivo signaling function of Nur77 thus appears to be mediated by dimers acting on a palindromic response element of unusual spacing between its half-sites. This mechanism may represent the biologically relevant paradigm of action for this subfamily of orphan nuclear receptors.