Nuclear Dbf2-related protein kinases (NDRs) in isolated cardiac myocytes and the myocardium: activation by cellular stresses and by phosphoprotein serine-/threonine-phosphatase inhibitors.
The nuclear Dbf2-related protein kinases 1 and 2 (NDR1/2) are closely-related AGC family kinases that are strongly conserved through evolution. In mammals, they are activated inter alia by phosphorylation of an hydrophobic domain threonine-residue [NDR1(Thr-444)/NDR2(Thr-442)] by an extrinsic protein kinase followed by autophosphorylation of a catalytic domain serine-residue [NDR1(Ser-281)/NDR2(Ser-282)]. We examined NDR1/2 expression and regulation in primary cultures of neonatal rat cardiac myocytes and in perfused adult rat hearts. In myocytes, transcripts for NDR2, but not NDR1, were induced by the hypertrophic agonist, endothelin-1. NDR1(Thr-444) and NDR2(Thr-442) were rapidly phosphorylated (maximal in 15-30 min) in myocytes exposed to some phosphoprotein Ser-/Thr-phosphatase 1/2 inhibitors (calyculin A, okadaic acid) and, to a lesser extent, by hyperosmotic shock, low concentrations of H(2)O(2), or chelerythrine. In myocytes adenovirally-transduced to express FLAG-NDR2 (which exhibited a mainly-cytoplasmic localisation), the same agents increased FLAG-NDR2 activity as assessed by in vitro protein kinase assays, indicative of FLAG-NDR2(Ser-282/Thr-442) phosphorylation. Calyculin A-induced phosphorylation of NDR1(Thr-444)/NDR2(Thr-442) and activation of FLAG-NDR2 were inhibited by staurosporine, but not by other protein kinase inhibitors tested. In ex vivo rat hearts, NDR1(Thr-444)/NDR2(Thr-442) were phosphorylated in response to ischaemia-reperfusion or calyculin A. From a pathological viewpoint, we conclude that activities of NDR1 and NDR2 are responsive to cytotoxic stresses in heart preparations and this may represent a previously-unidentified response to myocardial ischaemia in vivo.
National Heart and Lung Institute (NHLI) Division, Faculty of Medicine, Imperial College London, Flowers Building, Armstrong Road, London SW7 2AZ, UK.
Stephen J
SJ
Fuller
Sampsa
S
Pikkarainen
El Li
el L
Tham
Timothy E
TE
Cullingford
Jeffery D
JD
Molkentin
Hauke
H
Cornils
Alexander
A
Hergovich
Brian A
BA
Hemmings
Angela
A
Clerk
Peter H
PH
Sugden
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D008387:Marine Toxins; D015428:Myocardial Reperfusion Injury / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D032383:Myocytes, Cardiac / Q000201:enzymology*; D009997:Osmotic Pressure; D010080:Oxazoles / Q000494:pharmacology; D018384:Oxidative Stress; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013912:Threonine / Q000378:metabolism; D000099259:N-myc Downstream-Regulated Gene 1 Protein
D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D010749:Phosphoprotein Phosphatases; D017346:Protein Serine-Threonine Kinases; D013912:Threonine; C059041:calyculin A; C531302:Ndr2 protein, rat; C531303:Ndrg1 protein, rat; D000099259:N-myc Downstream-Regulated Gene 1 Protein