The protein kinase KSR interacts with 14-3-3 protein and Raf.
BACKGROUND
KSR (kinase suppressor of Ras) is a recently identified putative protein kinase that positively mediates the Ras signaling pathway in the invertebrates Caenorhabditis elegans and Drosophila melanogaster. The function of vertebrate KSR is not well characterized biochemically or biologically.


RESULTS
We examined the physiological role of KSR in vertebrate signal transduction using Xenopus laevis oocytes. Overexpression of KSR, in combination with overexpression of the intracellular dimeric protein 14-3-3, induced Xenopus oocyte meiotic maturation and cdc2 kinase activation; the effect of KSR and 14-3-3 on oocyte maturation was blocked by co-expression of dominant-negative Raf-1. We noted that KSR contains multiple potential binding sites for 14-3-3, and we used the yeast two-hybrid system and co-immunoprecipitation experiments to show that KSR can bind to 14-3-3. Furthermore, we demonstrated that KSR can form a complex with Raf kinase both in vitro and in cultured cells. Cell fractionation studies revealed that KSR formed a complex with 14-3-3 in both the membrane and cytoplasmic fractions of cell lysates; however, KSR only formed a complex with Raf-1 in the membrane fraction.


CONCLUSIONS
Our finding suggest that KSR, 14-3-3 and Raf form an oligomeric signaling complex and that KSR positively regulates the Ras signaling pathway in vertebrate organisms.
Xing
H
H
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Kornfeld
K
K
Muslin
A J
AJ
D048948:14-3-3 Proteins; D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D005260:Female; D006801:Humans; D015151:Immunoblotting; D008540:Meiosis; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D014446:Tyrosine 3-Monooxygenase; D014982:Xenopus laevis
D048948:14-3-3 Proteins; D029742:Caenorhabditis elegans Proteins; D010446:Peptide Fragments; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C106207:par-5 protein, C elegans; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; C097199:KSR-1 protein kinase; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases