Biochemical characterization of the Cool (Cloned-out-of-Library)/Pix (Pak-interactive exchange factor) proteins.
The Cool (Cloned out of Library)/Pix (Pak interactive exchange factor) proteins have been implicated in a diversity of biological activities, ranging from pathways initiated by growth factors and chemoattractants to X-linked mental retardation. Initially discovered through yeast two-hybrid and biochemical analyses as binding partners for the Cdc42/Rac-target/effector, Pak (p21 activated kinase), the sequences for the Cool/Pix proteins revealed a DH (Dbl homology) domain. Because the DH domain is the limit functional unit for stimulating guanine nucleotide exchange on Rho family GTP-binding proteins, it was assumed that the Cool/Pix proteins would act as guanine nucleotide exchange factors (GEFs) for the Rho proteins. Of the three known isoforms, (p50Cool-1, p85Cool-1/beta-Pix, and 90Cool-2/alpha-Pix), only Cool-2/alpha-Pix has exhibited significant GEF activity. A number of experimental techniques have been used to characterize Cool-2, and in vitro analysis has revealed that its GEF activity is under tight control through intramolecular interactions involving several binding partners. Here we describe the biochemical methods used to study the Cool/Pix proteins and, in particular, the regulation of the GEF activity of Cool-2/alpha-Pix.
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA.
Daniel
D
Baird
Qiyu
Q
Feng
Richard A
RA
Cerione
D000818:Animals; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D064067:Rho Guanine Nucleotide Exchange Factors; D018411:Spodoptera; D020764:cdc42 GTP-Binding Protein / Q000096:biosynthesis / Q000302:isolation & purification; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D011994:Recombinant Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins