Serine 68 Phospholemman Phosphorylation during Forskolin-Induced Swine Carotid Artery Relaxation

Abstract

Background: Phospholemman (PLM) is an abundant phosphoprotein in the plasma membrane of cardiac, skeletal and smooth muscle. It is a member of the FXYD family of proteins that bind to and regulate the Na,K-ATPase. Protein kinase A (PKA) is known to phosphorylate PLM on serine 68 (S68), although the functional effect of S68 PLM phosphorylation is unclear. We therefore evaluated S68 PLM phosphorylation in swine carotid arteries. Methods: Two anti-PLM antibodies, one to S68 phosphorylated PLM and one to unphosphorylated PLM, were made to PLM peptides in rabbits and tested with purified PLM and PKA-treated PLM. Swine carotid arteries were mounted isometrically, contracted, relaxed with forskolin and then homogenized. Proteins were separated on SDS gels and the intensity of immunoreactivity to the two PLM antibodies determined on immunoblots. Results: The antipeptide antibody ‘C2’ primarily reacted with unphosphorylated PLM, and the antipeptide antibody ‘CP68’ detected S68 PLM phosphorylation. Histamine stimulation of intact swine carotid artery induced a contraction, increased the CP68 PLM antibody signal and reduced the C2 PLM antibody signal. High extracellular [K⁺] depolarization induced a contraction without altering the C2 or CP68 PLM signal. Forskolin-induced relaxation of histamine or extracellular [K⁺] contracted arteries correlated with an increased CP68 signal. Nitroglycerin-induced relaxation was not associated with changes in the C2 or CP68 PLM signal. Conclusions: These data suggest that a contractile agonist increased S68 PLM phosphorylation. Agents that increase [cAMP], but not agents that increase [cGMP], increased S68 PLM phosphorylation. S68 PLM phosphorylation may be involved in cAMP-dependent regulation of smooth muscle force.

Protein kinase Cα activity is important for contraction-induced FXYD1 phosphorylation in skeletal muscle

“…The apparent unchanged phosphorylation of FXYD1ser68 in human skeletal muscles, as indicated by application of the AB_FXYD1ser68 (Fig. 5A), is likely a result of phosphorylation at multiple sites because phosphorylation of FXYD1 at multiple sites induced by incubation with histamine and PKC has been shown to reduce the binding of AB_FXYD1ser68 compared with selective phosphorylation of FXYD1 at ser68 induced by incubation with forskolin and PKA (8,26). Previous findings in rat soleus and mixed thigh skeletal muscles of unchanged FXYD1 ser68 phosphorylation during contractions using AB_FXYD1ser68 (24) is, therefore, also likely to be a result of FXYD1 phosphorylation at multiple residues.…”

“…Two polyclonal rabbit antibodies were used to determine contraction-induced changes in FXYD1 phosphorylation (kindly provided by Dr. J. Randall Moorman, University of Virginia, and Dr. D. Bers, Loyola University, respectively). These antibodies were developed to recognize either unphosphorylated FXYD1 proteins (AB_FXYD1, originally denoted as C2) (35) or FXYD1 proteins phosphorylated at serine 68 (AB_FXYD1ser68, originally denoted as CP68) (26). To verify that FXYD1 phosphorylation status could be determined in human skeletal muscle, phosphorylation was either reduced by extraction without phosphatase (PPase) inhibitors followed by incubation with PPase (-PPase; New England BioLabs) or preserved by extraction with PPase inhibitors and afterward not incubated with PPase, as described previously (29).…”

Characterization of the phospholemman knockout mouse heart: depressed left ventricular function with increased Na-K-ATPase activity

“…of Iowa Hybridoma Bank), ␣2-subunit (06-168, Upstate), ␣3-subunit (SA247, Affiniti Research Products), total ␣-subunit (␣5, Univ. of Iowa Hybridoma Bank), ␤1-subunit (06-170, Upstate), ␤2-subunit (06-1171, Upstate), phospholemman (C2 antibody) (25), Na/Ca exchanger (MA3-926, Affinity Bioreagents), sarco(endo)plasmic reticulum Ca-ATPase (SERCA)2a (MA3-919, Affinity Bioreagents), phospholamban (05-205, Upstate), ␣1c dihydropyridine receptor (ACC-003, Alomone Labs) and ␣-myosin heavy chain (HV11, Univ. of Iowa Hybridoma Bank).…”

Phospholemman regulates cardiac Na+/Ca2+ exchanger by interacting with the exchanger's proximal linker domain

“…3A). When CP68, a polyclonal antibody specific for PLM phosphorylated at Ser 68 (31), was used, stimulation of PKA by forskolin or PKC by PMA resulted in expected increases in phosphorylation of Ser 68 (Fig. 3B).…”

“…NMR (10) and infrared spectroscopy (2) showed that the TM domain of PLM reconstituted in liposomes is an ␣-helix with a maximum tilt of 15-17°. Specifically, NMR spectroscopic studies of highly purified PLM in model micelles indicate that the molecule consists of four ␣-helices: H1 (residues [12][13][14][15][16][17] is in the extracellular NH 2 terminus, H2 (residues [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] is the main TM helix followed by the short H3 (residues 39 -45), and H4 (residues 60 -68) in the COOH terminus is connected to H3 by a flexible linker (36). PKA phosphorylates Ser 68 , whereas PKC phosphorylates Ser 63 and Ser 68 , of PLM (37).…”