Multiplicity of the β Form of the cAMP-dependent Protein Kinase Inhibitor Protein Generated by Post-translational Modification and Alternate Translational Initiation

Abstract

Two distinct species of the thermostable inhibitor of the cAMP-dependent protein kinase, PKI␣ and PKI␤, exist that are the products of separate genes. The PKI␤ form, as first isolated from rat testis, is a 70-amino acid protein, but the genomic sequence suggested that an alternate form might exist, arising as a consequence of alternate translational initiation. This species, now termed PKI␤-78, has been synthesized by bacterial expression, demonstrated to be equipotent with PKI␤-70, and also now demonstrated to occur in vivo. By Western blot analyses, six additional species of PKI␤ are also evident in tissues. Two of these represent the phospho forms of PKI␤-78 and PKI␤-70. The other four represent phospho and dephospho forms of two higher molecular mass PKI␤ species. These latter forms are currently termed PKI␤-X and PKI␤-Y, awaiting the full elucidation of their molecular identity. In adult rat testis and cerebellum, PKI␤-70, PKI␤-X, and PKI␤-Y constitute 39, 23, and 32% and 15, 29, and 54% of the total tissue levels, respectively. In adult rat testis, 35-42% of each of these three species is present as a monophospho form, whereas no phosphorylation of them is evident in cerebellum. PKI␤-78 is present at much lower levels in both rat testis and cerebellum (ϳ6 and 2% of the total, respectively) and almost entirely as a monophospho species. PKI␤-78, like PKI␤-70, is a high affinity and specific inhibitor of the cAMP-dependent protein kinase. PKI␤-Y and PKI␤-X, in contrast, also significantly inhibit the cGMP-dependent protein kinase.The cAMP-dependent protein kinase (PKA) 1 plays a central role in the signal transduction of many hormones. The thermostable protein kinase inhibitor (PKI) is a highly specific competitive inhibitor of this enzyme that binds with high affinity to the protein substrate-binding site of the kinase (1). Its role as a regulator of PKA remains to be fully identified and appears to include not only the reduction of protein kinase activity, but also the trafficking of the protein kinase catalytic subunit between subcellular sites (2-4). Two genetically distinct forms of PKI (␣ and ␤) have been shown to be present in many tissues (5-9). Both forms act as specific pseudosubstrate inhibitors of PKA, have similar K i values in the subnanomolar range, and are likely both involved in intracellular trafficking (4). PKI␣ and PKI␤ share ϳ40% identity at the amino acid level (5), share very closely the same recognition determinants for the catalytic subunit of PKA (5, 10) and for nuclear export (9), but are notably distinct in the C-terminal half of the molecule. PKI␣ and PKI␤ each have a distinct tissue distribution (6).The PKI␤ form was first identified in rat testis (5) in a follow-up of observations made earlier by Means and co-workers (11, 12). The PKI␤ form purified was one of what appeared to be multiple forms of the protein present in testis, as was apparent from the DEAE chromatographic profile of the partially purified protein. The form purified was shown by protein sequencing and amino acid...

Specific Testicular Cellular Localization and Hormonal Regulation of the PKIα and PKIβ Isoforms of the Inhibitor Protein of the cAMP-dependent Protein Kinase

“…Western Blot Analyses of PKI Isoforms-The procedures for Western blot analyses were identical to those used in the accompanying paper (22). Unless indicated otherwise, powdered frozen tissue was extracted in 1 mM EDTA, pH 7.0, containing 1.6 g/ml aprotinin, 2 mM benzamidine, 0.1 mM TPCK, 1 M leupeptin, and 0.5 mM (2-aminoethyl)benzenesulfonyl fluoride and heated-treated for 10 min at 100°C, and insoluble material was removed by centrifugation.…”

“…We now observe with PKI␤ an intricate pattern of expression, especially evident by the profile of forms that evolve with germ cell maturation. These forms arise as a consequence of covalent modification and alternate translation (22). Left to be unraveled is the unique function/activity that each of the different PKI␤ forms manifests, and only from that knowledge will an understanding be derived of why the transitions among the forms may be important for the process of germ cell maturation.…”

“…1, a and c). (The designation used for the PKI␤ forms, as based upon their currently established characteristics (22), is depicted in Fig. 1d).…”

“…A high signal Using animals of the indicated ages, rat testis extracts were prepared and Western blot analyses of PKI isoforms were undertaken as described under "Experimental Procedures" and in Ref. 22. a and c, one-dimensional (SDS) and two-dimensional (isoelectric focusing and SDS) electrophoretic separation, respectively, with detection using anti-PKI␤- (5-22) level of expressed PKI␤ mRNA (Fig.…”

“…A complex pattern of PKI isoforms is also beginning to emerge. It is now established that the PKI isoforms, as first studied in skeletal muscle (17) and testis (18,19), arise from two distinct genes (20,21) and furthermore that at least eight forms of PKI␤ exist that differ due to at least post-translational modification and alternate translational initiation (22,23). In this report, we demonstrate that the PKI␣ and PKI␤ isoforms are differentially localized to Sertoli and germ cells, respectively; that there is a progressive change in PKI␤ isoform formation with germ cell development; that testis PKI␣ and PKI␤ are both under hormonal/developmental regulation; and that Sertoli cell-germ cell communication likely plays an important role in the regulation of PKI␤ expression.…”