Regulation of WNK1 by an autoinhibitory domain and autophosphorylation.
WNK family protein kinases are large enzymes that contain the catalytic lysine in a unique position compared with all other protein kinases. These enzymes have been linked to a genetically defined form of hypertension. In this study we introduced mutations to test hypotheses about the position of the catalytic lysine, and we examined mechanisms involved in the regulation of WNK1 activity. Through the analysis of enzyme fragments and sequence alignments, we have identified an autoinhibitory domain of WNK1. This isolated domain, conserved in all four WNKs, suppressed the activity of the WNK1 kinase domain. Mutation of two key residues in this autoinhibitory domain attenuated its ability to inhibit WNK kinase activity. Consistent with these results, the same mutations in a WNK1 fragment that contain the autoinhibitory domain increased its kinase activity. We also found that WNK1 expressed in bacteria is autophosphorylated; autophosphorylation on serine 382 in the activation loop is required for its activity.
Xu
Bing-e
BE
Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas 75390-9041, USA.
Min
Xiaoshan
X
Stippec
Steve
S
Lee
Byung-Hoon
BH
Goldsmith
Elizabeth J
EJ
Cobb
Melanie H
MH
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002460:Cell Line; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008239:Lysine / Q000378:metabolism; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000076204:WNK Lysine-Deficient Protein Kinase 1
D047908:Intracellular Signaling Peptides and Proteins; D015778:Minor Histocompatibility Antigens; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D000076204:WNK Lysine-Deficient Protein Kinase 1; C498743:WNK1 protein, human; D008239:Lysine