Prigmore, Elena; (1999) Rho family binding proteins in human neutrophils. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Rho family proteins mediate diverse cellular functions including regulation of the actin cytoskeleton, transcriptional activation, and the NADPH oxidase of phagocytic leukocytes. To understand the molecular mechanisms by which Rho family proteins act, Cdc42Hs and Rac1 interacting proteins were identified in human neutrophils. Purification of the major Cdc42Hs binding activity from neutrophil cytosol demonstrated the presence of distinct binding proteins at approximately 62, 65, and 68 kDa, which interacted with Cdc42Hs and Rac1 in a GTP-dependent manner. Purified protein fractions contained three Ste20 immunoreactive proteins, and included a p21-activated kinase (PAK) with similarity to brain enriched α-PAK, suggesting the existence of multiple isoforms of PAK in neutrophils. Cdc42Hs and Rac1 interacting proteins were also identified in membrane-cytoskeletal fractions and with the Arp2/3 complex. In the latter case these proteins included a β-PAK-like protein and p57 the human coronin-like protein. Rac1 interacted directly with the NADPH oxidase component p67 phox in a GTP- dependent manner, but not with the other components, p47phox, p40phox or the cytochrome b. Rac1 and Rac2 bound amino acid residues 170-199 of p67phox. Recombinant β-PAK was found to phosphorylate p67phox in vitro. Potential PAK phosphorylation sites were located close to the Rac binding and polyproline domains. Deletion of the C-terminal (amino acids 239-526), the C-terminal SH3 domain (amino acids 461-526) or the polyproline domain (amino acids 226-236) of p67phox stimulated Rac1 binding ~8-fold and also β-PAK phosphorylation. These observations suggest that an intramolecular SH3-polyproline domain interaction may exist within p67phox and that the Rac1 and β-PAK-interacting sites in p67phox are cryptic. This study identifies three p21 binding partners in human neutrophils, PAK, p67phox and p57. Characterisation of these target proteins will be important for understanding the signal transduction pathways through which Rho family proteins carry out their cellular functions.

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