Targeting of alpha-kinase-anchoring protein (alpha KAP) to sarcoplasmic reticulum and nuclei of skeletal muscle.
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 Ca(2+) pumps, and junctional SR (jSR) membrane, enriched in ryanodine-sensitive Ca(2+)-release channels, and contains calsequestrin within its lumen. Protein phosphorylation mediated by the Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) has significant, distinct regulatory roles in both Ca(2+) uptake and Ca(2+) release. Kinase-anchoring proteins (KAPs) constitute a novel mechanism for achieving cell compartmentalization of effectors in phosphorylation pathways. Here, targeting of alpha KAP, a CaM kinase II-anchoring protein encoded within the alpha-CaM kinase II gene, was studied in transgenic skeletal muscle fibres of the adult rat soleus. The transgenes were epitope-tagged versions of alpha KAP and of a deletion mutant, allowing their specific immunodetection against the wild-type background. Our results show that alpha KAP is largely localized at the free SR and thus near the Ca(2+) pump, a protein that can be modulated by CaM kinase II phosphorylation. Only minor co-localization was observed with the jSR ryanodine-sensitive Ca(2+)-release channel, which is a potential CaM kinase II target. In non-muscle cells, recombinant alpha KAP is targeted to endoplasmic reticulum (ER). Both ER and SR targeting requires the N-terminal hydrophobic region of alpha KAP. 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.
Nori
Alessandra
A
Dipartimento di Scienze Biomediche Sperimentali dell'Università di Padova, viale G. Colombo 3, 35121 Padova, Italy.
Lin
Pei-Ju
PJ
Cassetti
Arianna
A
Villa
Antonello
A
Bayer
K-Ulrich
KU
Volpe
Pompeo
P
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology; D008297:Male; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D030781:Organisms, Genetically Modified; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D002352:Carrier Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases