Cyclic Adenosine Monophosphate-Dependent Phosphorylation of Mammalian Mitochondrial Proteins:  Enzyme and Substrate Characterization and Functional Role

Abstract

A study is presented on cyclic adenosine monophosphate- (cAMP-) dependent phosphorylation of mammalian mitochondrial proteins. Immunodetection with specific antibodies reveals the presence of the catalytic and the regulatory subunits of cAMP-dependent protein kinase (PKA) in the inner membrane and matrix of bovine heart mitochondria. The mitochondrial cAMP-dependent protein kinase phosphorylates mitochondrial proteins of 29, 18, and 6.5 kDa. With added histone as substrate, PKA exhibits affinities for ATP and cAMP and pH optimum comparable to those of the cytosolic PKA. Among the mitochondrial proteins phosphorylated by PKA, one is the nuclear-encoded (NDUFS4 gene) 18 kDa subunit of complex I, which has phosphorylation consensus sites in the C terminus and in the presequence. cAMP promotes phosphorylation of the 18 kDa subunit of complex I in myoblasts in culture and in their isolated mitoplast fraction. In both cases cAMP-dependent phosphorylation of the 18 kDa subunit of complex I is accompanied by enhancement of the activity of the complex. These results, and the finding of mutations in the NDUFS4 gene in patients with complex I deficiency, provide evidence showing that cAMP-dependent phosphorylation of the 18 kDa subunit of complex I plays a major role in the control of the mitochondrial respiratory activity.

cAMP controls oxygen metabolism in mammalian cells

“…3). In other cell lines, like mouse C2C12 myoblasts, cAMP stimulation of complex I activity was also observed in the exponential growth phase [9]. No significant changes in the activity of complex IV were, on the other hand, produced by serum-limitation of cell growth and subsequent treatment with cAMP (Fig.…”

“…The cAMP effects can be directly associated with phosphorylation of complex I subunits [9,[20][21][22] and or PKA-mediated changes in the redox [23]/nitrosylation [24] state of complex I subunits.…”

“…cAMP dependent protein kinase (PKA), which is present in different subcellular compartments, including mitochondria [9], activates the utilisation of glycogen and lipid energy stores as well as energy-requiring processes, like cell growth and development, neuronal activity, etc. [7].…”

A novel mitochondrial matrix serine/threonine protein phosphatase regulates the mitochondria permeability transition pore and is essential for cellular survival and development

“…In addition, a number of anchoring proteins for various protein kinases are also identified in mitochondria, including AKAP, PICK, Grb10, and Sab (Huang et al 1999;Nantel et al 1999;Alto et al 2002;Wiltshire et al 2002;Wang et al 2003). PKA activity targeted in the mitochondrial inner membrane and matrix is shown to activate Complex I of the respiratory chain in the bovine heart (Scacco et al 2000;Technikova-Dobrova et al 2001;Chen et al 2004). In addition, PKB/AKT activity leads to phosphorylation of the mitochondrial ATP synthase ␤ subunit and glycogen synthase kinase 3␤ (Gsk3␤), which in turn, inhibits pyruvate dehydrogenase activity (Bijur and Jope 2003).…”

Phosphorylation of B14.5a Subunit from Bovine Heart Complex I Identified by Titanium Dioxide Selective Enrichment and Shotgun Proteomics

“…In human (13) and mammalian cell cultures (7,14) cAMP-dependent in vitro phosphorylation of complex I subunits has been demonstrated and was associated with stimulation of the NADH:ubiquinone oxidoreductase activity of the complex. cAMP has also been found to reduce accumulation of oxygen free radicals from human and murine cells in culture (15,16). Changes in the reactive oxygen species (ROS) level appeared to be inversely related to cAMPdependent activation of complex I (17).…”