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10.1042/BJ20111879 http://scihub22266oqcxt.onion/10.1042/BJ20111879 22032326!3242505!22032326     free     free     free Deprecated: Implicit conversion from float 225.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 225.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 225.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 225.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 225.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 225.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 225.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 225.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 259.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534       Biochem+J 2012 ; 441 (1): 325-37 Nephropedia Template TP {{tp|p=22032326|t=2012. SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation |pdf=|usr=}}{{22032326}} gab.com Text SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation JO: Biochem J http://www.kidney.de/pget.php?&tw=1&pmid=22032326 #FOAvip Mutations in the WNK [with no lysine (K) kinase] family instigate hypertension and pain perception disorders. Of the four WNK isoforms, much of the focus has been on WNK1, which is activated in response to osmotic stress by phosphorylation of its T-loop residue (Ser382). WNK isoforms phosphorylate and activate the related SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) protein kinases. In the present study, we first describe the generation of double-knockin ES (embryonic stem) cells, where SPAK and OSR1 cannot be activated by WNK1. We establish that NKCC1 (Na+/K+/2Cl- co-transporter 1), a proposed target of the WNK pathway, is not phosphorylated or activated in a knockin that is deficient in SPAK/OSR1 activity. We also observe that activity of WNK1 and WNK3 are markedly elevated in the knockin cells, demonstrating that SPAK/OSR1 significantly influences WNK activity. Phosphorylation of another regulatory serine residue, Ser1261, in WNK1 is unaffected in knockin cells, indicating that this is not phosphorylated by SPAK/OSR1. We show that WNK isoforms interact via a C-terminal CCD (coiled-coil domain) and identify point mutations of conserved residues within this domain that ablate the ability of WNK isoforms to interact. Employing these mutants, we demonstrate that interaction of WNK isoforms is not essential for their T-loop phosphorylation and activation, at least for overexpressed WNK isoforms. Moreover, we finally establish that full-length WNK1, WNK2 and WNK3, but not WNK4, are capable of directly phosphorylating Ser382 of WNK1 in vitro. This supports the notion that T-loop phosphorylation of WNK isoforms is controlled by trans-autophosphorylation. These results provide novel insights into the WNK signal transduction pathway and provide genetic evidence confirming the essential role that SPAK/OSR1 play in controlling NKCC1 function. They also reveal a role in which the downstream SPAK/OSR1 enzymes markedly influence the activity of the upstream WNK activators. The knockin ES cells lacking SPAK/OSR1 activity will be useful in validating new targets of the WNK signalling pathway. Twit Text FOAVip SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation ????Biochem J http://www.kidney.de/pget.php?&tw=1&pmid=22032326 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=22032326 #FOAvip English Wikipedia <ref>{{Cite pmid|22032326}}</ref> SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation #MMPMID22032326 Thastrup JO; Rafiqi FH; Vitari AC; Pozo-Guisado E; Deak M; Mehellou Y; Alessi DR Biochem J 2012[Jan]; 441 (1): 325-37 PMID22032326show ga Mutations in the WNK [with no lysine (K) kinase] family instigate hypertension and pain perception disorders. Of the four WNK isoforms, much of the focus has been on WNK1, which is activated in response to osmotic stress by phosphorylation of its T-loop residue (Ser382). WNK isoforms phosphorylate and activate the related SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) protein kinases. In the present study, we first describe the generation of double-knockin ES (embryonic stem) cells, where SPAK and OSR1 cannot be activated by WNK1. We establish that NKCC1 (Na+/K+/2Cl- co-transporter 1), a proposed target of the WNK pathway, is not phosphorylated or activated in a knockin that is deficient in SPAK/OSR1 activity. We also observe that activity of WNK1 and WNK3 are markedly elevated in the knockin cells, demonstrating that SPAK/OSR1 significantly influences WNK activity. Phosphorylation of another regulatory serine residue, Ser1261, in WNK1 is unaffected in knockin cells, indicating that this is not phosphorylated by SPAK/OSR1. We show that WNK isoforms interact via a C-terminal CCD (coiled-coil domain) and identify point mutations of conserved residues within this domain that ablate the ability of WNK isoforms to interact. Employing these mutants, we demonstrate that interaction of WNK isoforms is not essential for their T-loop phosphorylation and activation, at least for overexpressed WNK isoforms. Moreover, we finally establish that full-length WNK1, WNK2 and WNK3, but not WNK4, are capable of directly phosphorylating Ser382 of WNK1 in vitro. This supports the notion that T-loop phosphorylation of WNK isoforms is controlled by trans-autophosphorylation. These results provide novel insights into the WNK signal transduction pathway and provide genetic evidence confirming the essential role that SPAK/OSR1 play in controlling NKCC1 function. They also reveal a role in which the downstream SPAK/OSR1 enzymes markedly influence the activity of the upstream WNK activators. The knockin ES cells lacking SPAK/OSR1 activity will be useful in validating new targets of the WNK signalling pathway. |Amino Acid Sequence[MESH] |Embryonic Stem Cells[MESH] |Gene Expression Regulation[MESH] |HEK293 Cells[MESH] |Humans[MESH] |Intracellular Signaling Peptides and Proteins/genetics/*metabolism[MESH] |Minor Histocompatibility Antigens[MESH] |Molecular Sequence Data[MESH] |Mutation[MESH] |Phosphorylation[MESH] |Protein Isoforms[MESH] |Protein Serine-Threonine Kinases/genetics/*metabolism[MESH] |Protein Structure, Tertiary[MESH] |Sodium-Potassium-Chloride Symporters/genetics/*metabolism[MESH] |Solute Carrier Family 12, Member 2[MESH]SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform int(epmc).pdf DeepDyve Pubget Overpricing