Identification of a Novel ERK 1/2-Interacting A-Kinase Anchoring Protein
Initially identified in Chlamydomonas, radial spoke protein 3 (RSP3) is one of at least twenty identified radial spoke structural components of motile cilia and is required for axonemal sliding and flagellar motility. The mammalian orthologs for this and other radial spoke proteins, however, remain to be identified and fully characterized. Mammalian RSP3 was found to interact with ERK2 through a yeast two-hybrid screen designed to identify interactors that have a higher affinity for the phosphorylated, active form of ERK2. Confirming this finding, the human homolog long form, RSP3H, co-immunoprecipitates with ERK1/2 in HEK293 cells. Human RSP3, and its larger alternative start site gene product, radial spoke protein 3 homolog (RSP3H), are phosphorylated by ERK1/2 on threonine 286 in vitro and in cells. RSP3/RSP3H are also phosphorylated in vitro by cAMP-dependent protein kinase (PKA). Additionally, we showed that human RSP3H functions as an A-kinase anchoring protein (AKAP), and its ability to bind to the regulatory subunits of PKA, RII and RII, is regulated by ERK1/2 activity and phosphorylation. Interestingly, expression analysis of mRNA suggests RSP3/RSP3H are also present in cells that are thought to contain a single primary cilium but not motile cilia. Immunofluorescence staining of primary cilia-containing cells indicates that RSP3/RSP3H localize to nuclear punctae, specifically promyelocytic leukemia (PML) bodies, suggesting a non-cilia related role for RSP3/RSP3H in these cells. Functionally, RSP3/RSP3H may localize ERK1/2 to a distinct site of action within the cell and serve as a point of convergence of cAMP-dependent and PKA-mediated influence upon ERK1/2 downstream signaling or vice versa. These data are the first to establish a connection between ERK1/2 and what was once ostensibly thought to only be a ciliary component as well as to identify a novel ERK1/2-interacting AKAP.
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