Analysis of sites phosphorylated on acetyl‐CoA carboxylase in response to insulin in isolated adipocytes

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Abstract
We have examined the sites phosphorylated on acetyl‐CoA carboxylase in response to insulin in isolated adipocytes. Two tryptic peptides derived from the enzyme become more radioactive after treatment of 32P‐labelled cells with insulin. One of these (T4a) accounts for a large part of the total increase in phosphate observed after insulin treatment, and comigrates with the pep tide containing the sites phosphorylated in vitro by casein kinase‐2. The other may correspond to the T site peptide originally described by Brownsey and Denton in 1982: labelling of this peptide is stimulated at least threefold by insulin treatment, but it is a minor phosphopeptide and, even after insulin treatment, accounts for only about 2.5% of the enzyme‐bound phosphate (equivalent to < 0.1 mol phosphate/mol 240‐kDa subunit). Two other major tryptic phosphopeptides (T1 and T4b) labelled in adipocytes do not change significantly in response to insulin, and comigrate with peptides containing sites phosphorylated in vitro by cyclic‐AMP‐dependent protein kinase and calmodulin‐dependent multiprotein kinase respectively. We have sequenced peptides T4a and T4b from acetyl‐CoA carboxylase derived from control and insulin‐treated adipocytes, and also after phosphorylation in vitro with casein kinase‐2 and the calmodulin‐dependent multiprotein kinase. The results show that T4a and T4b are forms of the same peptide containing phosphate groups on different serine residues: Phe‐Ile‐Ile‐Gly‐Ser4‐Val‐Ser5‐Gln‐Asp‐Asn‐Ser6‐Glu‐Asp‐Glu‐Ile‐Ser‐Asn‐Leu‐. Site 5 was phosphorylated by the calmodulin‐dependent protein kinase and site 6 by casein kinase‐2. Migration in the T4a position was exclusively associated with phosphorylation in site 6, irrespective of the presence of phosphate in sites 4 and 5. Sites 5 and 6 were partially phosphorylated in control adipocytes, and there were also small amounts of phosphate in site 4. On stimulation with insulin, phosphorylation appeared to occur primarily at site 6, thus accounting for the increase in 32P‐labelling of T4a. We were unable to isolate sufficient quantities of the other insulin‐sensitive peptide to determine its sequence. Our results are consistent with the idea that insulin activates either casein kinase‐2, or a protein kinase which has the same specificity as casein kinase‐2. The function of this modification is not clear, since phosphorylation by casein kinase‐2 has no direct effect on acetyl‐CoA carboxylase activity.