Identification and Characterization of a Novel Gene, dapr, Involved in Skeletal Muscle Differentiation and Protein Kinase B Signaling

Abstract

The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been associated with a variety of cellular functions ranging from cell cycle regulation to tissue development. Although years of research have extensively characterized this signaling pathway, little is known as to how specific cellular events are coordinated by its activation. Here we demonstrate that Dapr (differentiation-associated protein), a novel protein, appears to focus one aspect of this pathway by acting as a putative scaffold protein during skeletal muscle differentiation. We present for the first time a description of this protein using in silico analysis. dapr was discovered through a previous study employing chromatin immunoprecipitation and CpG microarray analysis experiments as being regulated by myocyte-enhancing factor 2, a key transcription factor involved in the differentiation of skeletal muscle tissue. In this study we show that during the course of differentiation, Dapr binds to the PI3K signaling pathway member protein kinase B (PKB). In C 2 C 12 myoblast cells before differentiation Dapr is localized to the cytosol, migrating with PKB to the membrane after initiation of muscle differentiation. Knockdown of Dapr by RNAi resulted in inhibition of myotube formation. Our findings indicate that Dapr is a key component required by myoblasts for orchestrating their differentiation during myogenesis. Furthermore, it appears that Dapr is involved in the PI3K signaling cascade, potentially acting as a scaffold protein for PKB and coordinating its compartmentalization during differentiation. In the transformation of myoblasts into myotubes, PI3K2 signaling is one of the key pathways (1). Activation of this pathway typically begins with signaling through the activated surface receptor by insulin growth factor 1 (2). Downstream, PI3K-targeted phosphorylation produces the lipid products phosphatidylinositol 3,4-diphosphate and phosphatidylinositol 3,4,5-trisphosphate. These phosphoinositides provide membrane anchoring points for pleckstrin homology domain-containing proteins with binding motifs to them. Protein kinase B (PKB, also known as AKT) is one such protein (3). The targeted localization of PKB and other kinases to the plasma membrane is an important part of their activation (4, 5). Indeed, the compartmentalization of kinases within the cell has come to be known as a key regulatory mechanism of signaling cascades. A kinase-associated protein (6) is an example of one of the first proteins identified to function in this manner. Not only does A kinase-associated protein localize kinases to specific parts of the cell, it also brings proteins together within proximity of one another; in this case, protein phosphatase 2 and protein kinase A (7).Among its many important roles as a signal transducer, including insulin-mediated signaling, PKB has also been shown to play a pivotal role in cellular differentiation (1,8,9). Its role in the development of myotubes has been well established. Initial work demonstrated that PI3K inhibition causing ...

Proto-oncogenic H-Ras, K-Ras, and N-Ras are involved in muscle differentiation via phosphatidylinositol 3-kinase

“…The scaffold protein could operate selectively on activated c-Ras bound to the PI3K p110 subunit, but not bound to c-Raf. Similarly, it was reported that Dapr (differentiation-associated protein), a novel protein, acted as a scaffold protein for Akt during muscle differentiation [41]. These reports along with our data indicate that cells may have devised multiple mechanisms to activate PI3K with concurrent Raf inactivation downstream of c-Ras during myogenic differentiation.…”