Two splice variants of CaMKII-anchoring protein are present in the sarcoplasmic reticulum of rabbit fast-twitch muscle.
Anchoring protein alphaKAP targets calmodulin kinase II (CaMKII) to the sarcoplasmic reticulum (SR), and in the rabbit is a substrate of CaMKII itself in fast-twitch, but not in slow-twitch muscle. This work was aimed at elucidating the molecular basis for differential phosphorylation of alphaKAP. Here we show that two, immunologically related, size forms (23 and 21 kDa) of alphaKAP are present in fast-twitch muscle SR in a 3:1 stoichiometry. Phosphorylation experiments identified the shorter form as the CaMKII specific substrate. Both forms are shown to be stably integrated into the holoenzyme. Two splice variants of alphaKAP were found in rabbit fast-twitch muscle and only one in slow-twitch muscle, using RT-PCR. Mobilities on SDS-PAGE are those expected. The shorter splice variants lacks the 33-nucleotide sequence inserted by alternative splicing present in full-length alphaKAP, akin to differences between variants A and B of brain alphaCaMKII. The absence of the 11-amino acid sequence creates a novel CaMKII phosphorylation site. Taken together our results show that alternative splicing regulates alphaKAP phosphorylation in a fiber-type specific manner.
Damiani
Ernesto
E
NRC Unit for Muscle Biology and Physiopathology, Department of Experimental Biomedical Sciences, University of Padova, viale Giuseppe Colombo 3, Padova 35121, Italy.
Sacchetto
Roberta
R
Salviati
Leonardo
L
Margreth
Alfredo
A
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D008297:Male; D008969:Molecular Sequence Data; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity
D017931:DNA Primers; D017871:Calcium-Calmodulin-Dependent Protein Kinases