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Identification of a neuroregulated phosphoprotein in skeletal muscle as the regulatory subunit of cyclic AMP‐dependent protein kinase II
Author(s) -
Squinto S. P.,
McLane J. A.,
Held I. R.
Publication year - 1985
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.490140207
Subject(s) - phosphorylation , phosphoprotein , protein kinase a , proteolysis , trypsin , autophosphorylation , biochemistry , microbiology and biotechnology , protein subunit , protein phosphorylation , biology , denervation , chemistry , endocrinology , enzyme , gene
When soluble proteins in cytosolic fractions of rat soleus muscles are 32 P‐phosphorylated in vitro by an ATP:protein phosphotransferase reaction, the major substrate is a 56‐kilodalton (56K) protein. As we have also reported previously, the onset and development of increased 32 P‐phosphorylation of this 56K protein, which are observed after the soleus is denervated, temporally correlate with the denervation period and length of the distal nerve stump [Held et al, 1983]. Conclusive evidence which identifies this neuroregulated muscle protein as the regulatory subunit of cyclic AMP‐dependent protein kinase type II (R‐II) is presented in this paper. The 56K soleus protein and purified bovine heart R‐II were 32 P‐phosphorylated and subjected to limited proteolysis with bovine pancreas trypsin. After resolution of the generated 32 P‐phosphopeptides by SDS slab PAGE and visualization by autoradiography, no tryptic products were observed from the 56K soleus protein which were not also produced by proteolysis of the purified R‐II. These tryptic phosphopeptides included 39, 16.5, and 12K fragments which retained the autophosphorylation site of R‐II. After denervation, the 32 P‐phosphorylation of the 56K soleus protein and of the 39K tryptic peptide product were comparably increased. The identification of the neuroregulated 56K soleus protein as R‐II was also confirmed by Western blotting with a specific anti‐R‐II sera. Taken together, our results demonstrate that the previously observed neuroregulation of the 32 P‐phosphorylation of the 56K soleus protein is identifiable with some alteration which affects the intramolecular 32 P‐autophosphorylation of R‐II.