The sarcoplasmic reticulum (SR) plays a key role in excitation/contraction coupling of skeletal muscle. The SR is composed of two continuous yet heterogeneous membrane compartments, the free or longitudinal SR and cisternal SR. Cisternal SR is made up of free SR membrane, enriched in Ca2+ pumps, and junctional SR (jSR) membrane, enriched in ryanodine-sensitive Ca2+-release channels, and contains calsequestrin within its lumen. Protein phosphorylation mediated by the Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) has significant, distinct regulatory roles in both Ca2+ uptake and Ca2+ release. Kinase-anchoring proteins (KAPs) constitute a novel mechanism for achieving cell compartmentalization of effectors in phosphorylation pathways. Here, targeting of αKAP, a CaM kinase II-anchoring protein encoded within the α-CaM kinase II gene, was studied in transgenic skeletal muscle fibres of the adult rat soleus. The transgenes were epitope-tagged versions of αKAP and of a deletion mutant, allowing their specific immunodetection against the wild-type background. Our results show that αKAP is largely localized at the free SR and thus near the Ca2+ pump, a protein that can be modulated by CaM kinase II phosphorylation. Only minor co-localization was observed with the jSR ryanodine-sensitive Ca2+-release channel, which is a potential CaM kinase II target. In non-muscle cells, recombinant αKAP is targeted to endoplasmic reticulum (ER). Both ER and SR targeting requires the N-terminal hydrophobic region of αKAP. An unexpected additional specific localization that does not require the N-terminus was found in the nucleus, providing a first clue of how CaM kinase II can fulfil its nuclear functions in skeletal muscle.
Abbreviations used: CaM kinase II, Ca2+/calmodulin-dependent protein kinase II; CR, calreticulin; CS, calsequestrin; ER, endoplasmic reticulum; HA, haemagglutinin A; jSR, junctional SR; KAP, kinase-anchoring protein; NLS, nuclear localization signal; RyR, ryanodine receptor; SERCA, sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum.