THE MULTIPHOSPHORYLATION DOMAIN OF THE PHOSPHORYLASE-KINASE ALPHA(M) AND ALPHA(L) SUBUNITS IS A HOTSPOT OF DIFFERENTIAL MESSENGER-RNA PROCESSING AND OF MOLECULAR EVOLUTION

We have cloned and sequenced human cDNAs encoding the complete phospho rylase kinase alpha subunit muscle isoform (alpha(M)). The predicted p olypeptide is highly similar to the sequence known from rabbit muscle but lacks a major part of its multiphosphorylation domain, including t he main phosphorylation site for cAMP-dependent protein kinase (PKA). Analysis of this region by reverse-transcribed polymerase chain reacti on (RT-PCR) in several human and rabbit tissues demonstrates that it i s subject to elaborate differential mRNA splicing. Amino acids 1012-10 24 of the full-length rabbit sequence, including the major PKA phospho rylation site, and amino acids 1025-1041, which harbor at least one en dogenous phosphorylation site, can be deleted from the predicted polyp eptide individually or in combination. Molecules lacking one or both o f these segments constitute a major part of the alpha(M) subunit popul ation in many rabbit tissues and constitute the vast majority in all h uman tissues analyzed. Similar, tissue-dependent differential splicing events could be detected by RT-PCR in the human alpha subunit isoform from liver (alpha(L)). The expression of the differentially spliced a lpha(M) subtypes differs markedly between corresponding human and rabb it tissues. Sequence divergence in this region is particularly high, n ot only between the muscle and liver isoforms, but also between alpha( M) sequences from four different animal species. Moreover, a duplicati on of the exon encoding the main PKA phosphorylation site was discover ed in the mouse. Thus, the multiphosphorylation domain of the phosphor ylase kinase alpha subunit isoforms is subject to pronounced structura l variation not only between different tissues of one organism via dif ferential splicing, but also in the course of evolution.