Obscurins comprise a family of giant<br>(870- to 600-kDa) and small (250- to 55-kDa) proteins<br>that play important roles in myofibrillogenesis,<br>cytoskeletal organization, and cell adhesion and are<br>implicated in hypertrophic cardiomyopathy and tumorigenesis.<br>Giant obscurins are composed of tandem<br>structural and signaling motifs, including 2 serine/<br>threonine kinase domains, SK1 and SK2, present at the<br>COOH terminus of giant obscurin-B. Using biochemical<br>and cellular approaches, we show for the first time<br>that both SK1 and SK2 possess enzymatic activities and<br>undergo autophosphorylation. SK2 can phosphorylate<br>the cytoplasmic domain of N-cadherin, a major component<br>of adherens junctions, and SK1 can interact with<br>the extracellular domain of the 1-subunit of the Na/<br>K-ATPase, which also resides in adherens junctions.<br>Immunostaining of nonpermeabilized myofibers and<br>cardiocytes revealed that some obscurin kinase isoforms<br>localize extracellularly. Quantification of the<br>exofacial expression of obscurin kinase proteins indicated<br>that they occupy 16 and 5% of the sarcolemmal<br>surface in myofibers and cardiocytes, respectively.<br>Treatment of heart lysates with peptide-N-glycosidase F<br>revealed that while giant obscurin-B localizes intracellularly,<br>possessing dual kinase activity, a small obscurin<br>kinase isoform that contains SK1 localizes extracellularly,<br>where it undergoes N-glycosylation. Collectively,<br>our studies demonstrate that the obscurin kinase domains<br>are enzymatically active and may be involved in<br>the regulation of cell adhesion.