Identification of a novel phosphorylation site in protein phosphatase inhibitor-1 as a negative regulator of cardiac function.
Human and experimental heart failure is characterized by increases in type-1 protein phosphatase activity, which may be partially attributed to inactivation of its endogenous regulator, protein phosphatase inhibitor-1. Inhibitor-1 represents a nodal integrator of two major second messenger pathways, adenosine 3',5'-cyclic monophosphate (cAMP) and calcium, which mediate its phosphorylation at threonine 35 and serine 67, respectively. Here, using recombinant inhibitor-1 wild-type and mutated proteins, we identified a novel phosphorylation site in inhibitor-1, threonine 75. This phosphoamino acid was phosphorylated in vitro by protein kinase Calpha independently and to the same extent as serine 67, the previous protein kinase Calpha-identified site. Generation of specific antibodies for the phosphorylated and dephosphorylated threonine 75 revealed that this site is phosphorylated in rat and dog hearts. Adenoviral-mediated expression of the constitutively phosphorylated threonine 75 inhibitor-1 in isolated myocytes was associated with specific stimulation of type-1 protein phosphatase activity and marked inhibition of the sarcoplasmic calcium pump affinity for calcium, resulting in depressed contractility. Thus, phosphorylation of inhibitor-1 at threonine 75 represents a new mechanism of cardiac contractility regulation, partially through the alteration of sarcoplasmic reticulum calcium transport activity.
Rodriguez
Patricia
P
Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267, USA.
Mitton
Bryan
B
Waggoner
Jason R
JR
Kranias
Evangelia G
EG
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D006321:Heart / Q000502:physiology*; D006801:Humans; D008297:Male; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000737:chemistry / Q000378:metabolism; D054645:Protein Phosphatase 1; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D011506:Proteins; D051571:Protein Kinase C-alpha; C450539:PPP1R2C protein, human; D054645:Protein Phosphatase 1; D002118:Calcium