Solution structure and functional analysis of the cysteine-rich C1 domain of kinase suppressor of Ras (KSR).
Kinase suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to promote signal transmission from Raf-1 to MEK and MAPK. All KSR proteins contain a conserved cysteine-rich C1 domain, and studies have implicated this domain in the regulation of KSR1 subcellular localization and function. To further elucidate the biological role of the KSR1 C1 domain, we have determined its three-dimensional solution structure using nuclear magnetic resonance (NMR). We find that while the overall topology of the KSR1 C1 domain is similar to the C1 domains of Raf-1 and PKCgamma, the predicted ligand-binding region and the surface charge distribution are unique. Moreover, by generating chimeric proteins in which these domains have been swapped, we find that the C1 domains of Raf-1, PKCgamma, and KSR1 are not functionally interchangeable. The KSR1 C1 domain does not bind with high affinity or respond biologically to phorbol esters or ceramide, and it does not interact directly with Ras, indicating that the putative ligand(s) for the KSR1 C1 domain are distinct from those that interact with PKCgamma and Raf-1. In addition, our analysis of the chimeric proteins supports the model that Raf-1 is a ceramide-activated kinase and that its C1 domain is involved in the ceramide-mediated response. Finally, our findings demonstrate an absolute requirement of the KSR1 C1 domain in mediating the membrane localization of KSR1, a crucial feature of its scaffolding activity. Together, these results underscore the functional specificity of these important regulatory domains and demonstrate that the structural features of the C1 domains can provide valuable insight into their ligand-binding properties.
Zhou
Ming
M
Regulation of Cell Growth Laboratory, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702, USA.
Horita
David A
DA
Waugh
David S
DS
Byrd
R Andrew
RA
Morrison
Deborah K
DK
D016475:3T3 Cells; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002518:Ceramides / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D020935:MAP Kinase Signaling System; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009865:Oocytes / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D012996:Solutions; D014982:Xenopus laevis
D002518:Ceramides; D007527:Isoenzymes; D008024:Ligands; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D012996:Solutions; D011494:Protein Kinases; C097199:KSR-1 protein kinase; C087857:protein kinase C gamma; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D003545:Cysteine