Protein kinases represent a superfamily of over 400 members in 57 subfamilies that share a catalytic domain (12 subdomains) of 250-300 amino acids. Some of these residues are conserved and crucial for structure and function of the active site. A conserved lysine, in subdomain II of the catalytic core of all 390-protein kinases, anchors and orientates the a and b phosphoryl groups of ATP. The catalytic domain of WNK (With No K=Lysine) kinases contains all invariant residues, except for this lysine residue. Being specific for multicellular organisms, the number of WNK genes an organism reflects the organism's complexity. There are 4 (identified) WNK genes (WNK1, WNK2, WNK3 and WNK4) in human and rat, one in C. elegans and D. melanogaster. The hWNK kinases exhibit ~30% seq identity and 50% seq homology to human Mitogen Activated Protein kinases (MAPK) STK2, PAK2, MEKK3, PAK3 and Raf-1. The mutation in genes expressing WNK1 (chromosome 12p13.3) and WNK4 (chromosome 17q) causes Pseudohypoaldosteronism type II (PHAII), an autosomal disorder featuring hypertension, hyperkalemia (high serum K+ level), normal/elevated aldosterone level, increased renal salt absorption and impaired K+ and H+ excretion.

WNK1 (251 kDa; 2382aa, chromosome 12p13.3) is a Ser-Thr kinase in which the conserved catalytic lysine in subdomain II is replaced by a cysteine but another lysine in subdomain I confers the kinase activity. The hWNK1 is ~86% identical to rat WNK1 (230 kDa, 2126aa). The two transcripts of hWNK1 (8.5 and 10 kb) enzyme are expressed in most tissues but predominantly in kidney, heart and muscle and weakly in brain. In cells of these tissues, WNK1 is present throughout the cytoplasm. WNK1 localizes to the distal convoluted tubule (DCT), medullary and cortical collecting ducts (MCD and CCD), adjacent segments of the distal nephron that play a key role in salt, water, K+ and pH homeostasis. In the CD, Na+ is reabsorbed by the epithelial sodium channel (ENaC) providing the electrical diving force for K+ and H+ secretion. Considering the PHAII-features, WNK1 possibly, increases the transcellular or paracellular Cl- conductance in CD, thereby increasing salt reabsorption and intravascular volume, while concomitantly dissipating the K+ and H+ secretion.

WNK2 consists of a single polypeptide chain in which the catalytic domain is ~91% homologous to that of hWNK1. The conserved catalytic lysine in subdomain II is replaced by a cysteine but another lysine residue in the glycine ribbon of the subdomain I confers the kinase activity. Eleven out of 33 residues in the subdomains I and II of WNK3 are conserved in the 18 WNK-related kinases. The WNK2 gene (chromosome 9q22.3) is predominantly expressed in heart, brain and colon.

WNK3 (partial 1450-aa, chromosome Xp11.21-23) is a Ser-Thr kinase with a catalytic domain which is ~93% homologous to that of hWNK1. The conserved catalytic lysine in subdomain II is replaced by a cysteine but another lysine residue in the glycine ribbon of the subdomain I confers the WNK3 kinase activity. Eleven out of 33 residues in the subdomains I and II of WNK3 are conserved in the 18 WNK-related kinases. The WNK3 gene (chromosome Xp11.21-23) is predominantly expressed in brain.

WNK4 (human 1243-aa, 17q21) is a single polypeptide chain kinase in which the conserved catalytic lysine is replaced by a cysteine but another lysine residue in subdomain I confers the kinase activity. Sequence of WNK4 (1243aa) is 76% identical to WNK1 in kinase domain. WNK4 is expressed predominantly in kidney, colon and skin. WNK4 localize to the distal convoluted tubule (DCT) and cortical collecting duct (CCD), that play a key role in salt, water, K+ and pH homeostasis. WNK4 is present exclusively in the intracellular tight junctions in DCT and in both cytoplasm and tight junctions in CCD. In DCT, salt reabsorption is mediated by the electroneutral Na-Cl cotransporters. In the CD, Na+ is reabsorbed by the epithelial sodium channel (ENaC) providing the electrical diving force for K+ and H+ secretion. Considering the PHAII-features, WNK4 possibly, increases the chloride conductance in CD, thereby increasing salt reabsorption and intravascular volume, while concomitantly diminishing K+ and H+ secretion.

ADI has produced highly specific rabbit polyclonal antibodies to WNK1, WNK2, WNK3 and WNK4 using the protein-specific peptide sequences. These antibodies should aid the studies aimed at identifying WNK kinase signaling pathway for the development of anti-hypertensive diuretic drugs.

 

M= Mouse; R=Rat; H=Human; Ha=Hamster; Rb=Rabbit; B=Bovine; CT= near C-terminus; NT=near N-terminus; Internal=Middle of protein. *

m=mouse; r=rat; h=human; b=bovine; d=dog; ~CT or ~NT=near C or N-terminus. EC=Extracellular; CP=Cytoplasmic domain; Control peptides (unconjugated, free, antigenic peptides), because of their small size, are not recommended for Western. They should be used in ELISA/antibody blocking studies.

* Expected antibody crossreactivity information is mostly based upon high (>70%) sequence conservation of antigenic/control peptides in various species. When antibody crossreactivity has actually been experimentally confirmed in various species, it will be mentioned in the appropriate data sheets.

"Neat Antisera or antisera" are the unpurified antiserum and it is suitable for ELISA and Western.
"Affinity pure" IgG may be more suitable for immunohistochemical (IHC) applications and to reduce background in most immunological applications including ELISA and Western.
"Control peptides" can not be used for Western as they are very short peptides. They are intended for ELISA or antibody blocking studies to establish antibody specificity.
Western blot +ve protein controls, where available, are semi-pure, pure or recombinant proteins that are formulated in SDS-PAGE sample buffer. They are recommended to be used for Western (load 10 ul/lane) for visualization with antibodies.

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