In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na ؉ /H ؉ exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser 282 , which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser 273 or Ser 302 , cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain-and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser 282 . Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser 282 phosphorylation by RSK. Immunolabeling with a Ser(P) 282 antibody and confocal fluorescence microscopy showed RSKmediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser 282 phosphorylation in cMyBP-C, accompanied by significant reduction in Ca 2؉ sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser 22 /Ser 23 . The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser 273 and Ser 302 . We conclude that Ser 282 in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.The 90-kDa ribosomal S6 kinase (p90 RSK or RSK), 3 also known as the mitogen-activated protein kinase-activated protein kinase-1 (MAPKAP-K1) family, comprises three principal isoforms: RSK1 (encoded by the RPS6KA1 gene), RSK2 (RPS6KA3), and RSK3 (RPS6KA2) (1). An additional isoform, RSK4 (RPS6KA6), has also been identified, but evidence suggests that this isoform behaves in an atypical manner in terms of its expression and function (2). All four isoforms are characterized by the presence of two functionally distinct kinase domains, the N-terminal kinase (NTK) and the C-terminal kinase (CTK) (1). RSKs bind and are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) through a sequential process that involves ERK-mediated phosphorylation of the CTK, CTK-mediated autophosphorylation of the RSK hydrophobic motif to create a docking site for 3Ј-phosphoinositide-dependent kinase 1 (PDK1), and PDK1-mediated phosphorylation of the NTK (1). Although the only known substrate of the CTK is the RSK hydrophobic motif, multiple cellular substrates are phosphorylated by the NTK, a serine/ threonine kinase of the AGC kinase family (1). Consistent with this, RS...