Phosphorylation site-specific antibodies to cardiac phospholamban.

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The majority of Ca2+ required for cardiac contraction is supplied hy the sarcoplasmic reticulum (SR), an intracellular store. Ca2+ released during contraction is returned to the SR by the activity of the enzyme Ca2+-Mg2+ ATPase, thus facilitating muscle relaxation. The sequestration of Ca2+ by the calcium pump is regulated hy ii second cardiac protein phospholamban (PLB), which in response to p-adrenergic stimulation of the heart is phosphorylated at two sites, Ser16 by the CAMP protein dependent kinasc (c-PKA), and Thr17 by Ca2+/calmodulin (CaM) dependent kinase I I [ I ] . This leads to increased pump activity resulting in an ahbrcviation of the Ca2+ transient, increased loading o l the SR which produces a larger Ca2+ transient at the next contraction. The contrihution o l each site of phosphorylation o f PLB to this process remains unknown, although hoth appear important in vivo 121. I t has hcen suggested that each signalling cascade controls a distinct region 0 1 thc SR 131. In this study we have prepared antihodics wholly specific for each of the two phosphorylated l'omis of PLB, which we aim to use to study the spatial distrihution of site-specific phosphorylation in cardiac muscle cells. Rabbits were immuniscd with a peptide derived from the phosphorylation sequence (if PLB (RSAIRRASTIEY amide [4]) following phosphorylation of either Ser16 or ThrI7. The spccilicity of antibodies so produced was examined, firstly by considering the PLB phosphorylation-site specificity of each antihody and then hy examining the discrimination between PLB and other muscle phosphoproteins. Phospholanihan was presented to these antibodies in three forms, unphosphorylated, SerI6 and ThrI7 phosphorylated as descrihed elsewhere [ S ] . Antiserum PSI6 (produced by immunisation with Ser16 phosphorylated peptide) detected Ser16 phosphorylated PLB exclusively. Similarly, antiserum PT17 only detected Thr I7 phosphorylated PLB. Thus antihodies totally specific for each phosphorylated form of phospholamban were produced. Figure I indicates the specilicity of the PS-16 and PT-17 antibodies when presented with phosphorylated cardiac tissue honiogenatcs. Panel A shows an autoradiograph of the cardiac muscle phosphoproteins generated by CAMP and Ca2+/CaM dependent interventions in vitro. PLB represents one of a number of phosphoproteins, which is identified by its distinctive reduction to monomer on hoiling (arrow). The PSI6 antiserum proves to be completely specific for serine phosphorylated PLB in rat cardiac honiogenate, identifying no other components ( C ) . The hinding of the PS16 antiserum with serinc phosphoylated PLB is completely aholished when competed with the serine phosphorylated peptide (D). Thc PTI7 antiserum specifically recognises the threonine phosphorylated PLB (E), which is completely aholished when competed with the threoninc phosphorylated peptide (F). The PTI7 antiserum does crossreact with one additional component (4XkDa) which is unelfected hy competition with the threonine phosphorylated peptide and thus considered non-specific. We are currently raising monoclonal antibodies to hoth phosphorylation sites o f PLB in order to overcome the detection of this contminat ing component hy the PTI7 antihody. Fip. 1. Antibodv soecificitv in the Dresence of cardiac muscle protcins. Rat heart homogenates were phosphorylated in the presence of CAMP (lanes 1,2) or CaC12 and CaM (lanes 3,4) (7). Samples were solubilised at 30'C (lanes 1, 3) for 30 minutes o r at l00'C (lanes 2, 4) for 5 minutes in Laemmli sample buffer. 100pg protein loaded per track onto SDS-PAGE gels (7-20% linear acrylamide gradient). Proteins were transferred to PVDF membrane and probed with PS-16 (1 : lO 000; panel C); PS-16 ( I : 10 000) plus 1pM Ser16-phosphorylated peptide (panel D); PT-17 (I:S000; panel E); PT-17 (1:SOOO) plus 1pM Thr17 phosphopeptide (panel F). The immunohlots were developed using a peroxidase based chemilumincscent detection kit which we estimate is 100 times more sensitive than methoxy-naphthol based peroxidase detection. An autoradiograph (10 day exposure) oC a representative memhrane is shown (panel A). Arrows indicate phospholamban, pentameric and monomeric forms. Asterisk indicates 48kDa component recognised hy antiserum PT17.