Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overexpression in insect cells.

Members of the phosphoprotein phosphatase (PPP) family of protein serine/threonine phosphatases, including protein phosphatase (PP)1, PP2A and PP2B, share invariant active-site residues that are critical for catalytic function [Zhuo, Clemens, Stone and Dixon (1994) J. Biol. Chem. 269, 26234-26238]. Mutation of the active-site residues Asp(88) or His(118) within the human PP2A catalytic subunit (PP2Ac)alpha impaired catalytic activity in vitro; the D88N and H118N substitutions caused a 9- and 23-fold reduction in specific activity respectively, when compared with wild-type recombinant PP2Ac, indicating an important role for these residues in catalysis. Consistent with this, the D88N and H118N substituted forms failed to provide PP2A function in vivo, because, unlike wild-type human PP2Acalpha, neither substituted for the endogenous PP2Ac enzyme of budding yeast. Relative to wild-type PP2Ac, the active-site mutants were dramatically overexpressed in High Five insect cells using the baculovirus system. Milligram quantities of PP2Ac were purified from 1x10(9) High Five cells and the kinetic constants for dephosphorylation of the peptide RRA(pT)VA (single-letter amino-acid notation) by PP2Ac (K(m)=337.5 microM; k(cat)=170 s(-1)) and D88N (K(m)=58.4 microM; k(cat)=2 s(-1)) were determined. The results show that the substitution impairs catalysis severely without a significant effect on substrate binding, consistent with the PPP catalytic mechanism. Combination of the baculovirus and yeast systems provides a strategy whereby the structure-function of PP2Ac may be fully explored, a goal which has previously proven difficult, owing to the stringent auto-regulatory control of PP2Ac protein levels in vivo.

Domain swapping used to investigate the mechanism of protein kinase B regulation by 3-phosphoinositide-dependent protein kinase 1 and Ser473 kinase.

Protein kinase B (PKB or Akt), a downstream effector of phosphoinositide 3-kinase (PI 3-kinase), has been implicated in insulin signaling and cell survival. PKB is regulated by phosphorylation on Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and on Ser473 by an unidentified kinase. We have used chimeric molecules of PKB to define different steps in the activation mechanism. A chimera which allows inducible membrane translocation by lipid second messengers that activate in vivo protein kinase C and not PKB was created. Following membrane attachment, the PKB fusion protein was rapidly activated and phosphorylated at the two key regulatory sites, Ser473 and Thr308, in the absence of further cell stimulation. This finding indicated that both PDK1 and the Ser473 kinase may be localized at the membrane of unstimulated cells, which was confirmed for PDK1 by immunofluorescence studies. Significantly, PI 3-kinase inhibitors prevent the phosphorylation of both regulatory sites of the membrane-targeted PKB chimera. Furthermore, we show that PKB activated at the membrane was rapidly dephosphorylated following inhibition of PI 3-kinase, with Ser473 being a better substrate for protein phosphatase. Overall, the results demonstrate that PKB is stringently regulated by signaling pathways that control both phosphorylation/activation and dephosphorylation/inactivation of this pivotal protein kinase.

A human protein kinase Bgamma with regulatory phosphorylation sites in the activation loop and in the C-terminal hydrophobic domain.

We have cloned human protein kinase Bgamma (PKBgamma) and found that it contains two regulatory phosphorylation sites, Thr305 and Ser472, which correspond to Thr308 and Ser473 of PKBalpha. Thus it differs significantly from the previously published rat PKBgamma. We have also isolated a similar clone from a mouse cDNA library. In human tissues, PKBgamma is widely expressed as two transcripts. A mutational analysis of the two regulatory sites of human PKBgamma showed that phosphorylation of both sites, occurring in a phosphoinositide 3-kinase-dependent manner, is required for full activity. Our results suggest that the two phosphorylation sites act in concert to produce full activation of PKBgamma, similar to PKBalpha. This contrasts with rat PKBgamma, which is thought to be regulated by 3-phosphoinositide-dependent protein kinase 1 alone.

Protein kinase B/Akt is activated by polyomavirus middle-T antigen via a phosphatidylinositol 3-kinase-dependent mechanism.

The middle tumor antigen (middle-T) of mouse polyomavirus is responsible for the transforming potential of this virus. Middle-T has been shown to interact with a variety of cellular proteins known to mediate mitogenic signaling, like phosphatase-2A, Src family kinases, phosphatidylinositol 3-kinase (PI 3-kinase), the adapter protein SHC, phospholipase Cgamma-1 and 14-3-3 family proteins. Association with SHC and PI 3-kinase, respectively, stimulates two independent signaling pathways that are indispensible for viral oncogenicity. SHC activates the Ras/MAPK pathway via Grb2/SOS resulting in changes in early gene expression. The downstream targets of PI 3-kinase are less well studied but seem to impinge on serum response factor (SRF) which is also involved in regulating early gene expression. Recently, the protein kinase B/Akt (PKB/Akt) has been identified as a target of PI 3-kinase in receptor tyrosine kinase signaling. Here we show that PKB/Akt is a target of wild type middle-T, but not of mutants unable to activate PI 3-kinase. These data were confirmed using inhibitors of PI 3-kinase as well as dominant-negative alleles of the catalytic subunit of this lipid kinase. In addition, mutants of PKB/Akt lacking a pleckstrin homology domain and therefore unable to bind to D3 phospatidylinositides were not activated by middle-T. Taken together these data suggest that middle-T activates PKB/Akt in a PI 3-kinase-dependent manner. Furthermore, direct association with D3 phosphatidylinositides seems to be essential for activation of PKB/Akt.

Mitogenic activation, phosphorylation, and nuclear translocation of protein kinase Bbeta.

Protein kinase B (PKB) is a member of the second messenger-dependent family of serine/threonine kinases that has been implicated in signaling pathways downstream of growth factor receptor tyrosine kinases and phosphatidylinositol 3-kinase. Here we report the characterization of the human beta-isoform of PKB (PKBbeta). PKBbeta is ubiquitously expressed in a number of human tissues, with mRNA and protein levels elevated in heart, liver, skeletal muscle, and kidney. After transfection into HEK-293 or COS-1 cells, PKBbeta is activated 2- to 12-fold by mitogens and survival factors. Activation was due to phosphorylation on Thr-309 and Ser-474, which correspond to Thr-308 and Ser-473 implicated in the regulation of PKBalpha. Both phosphorylation and activation were prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Moreover, membrane-targeted PKBbeta was constitutively activated when overexpressed in HEK-293 cells. Although the specific activity of PKBbeta was lower than that of PKBalpha toward Crosstide as a substrate (23 nmol/min/mg compared with 178 nmol/min/mg for PKBalpha), both enzymes showed similar substrate specificities. Using confocal microscopy, we show that activation of PKBbeta results in its nuclear translocation within 20 to 30 min after stimulation. These observations provide evidence that PKBbeta undergoes nuclear translocation upon mitogenic activation and support a role for PKB in signaling from receptor tyrosine kinases to the nucleus through phosphatidylinositol 3-kinase.

Role of translocation in the activation and function of protein kinase B.

We have investigated the role of subcellular localization in the regulation of protein kinase B (PKB) activation. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N terminus of protein kinase B to alter its subcellular location. Myristoylated/palmitylated (m/p)-PKBalpha was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKBalpha was 60-fold higher compared with the unstimulated wild-type enzyme, and could not be stimulated further by growth factors or phosphatase inhibitors. In vivo 32P labeling and mutagenesis demonstrated that m/p-PKBalpha activity was due to phosphorylation on Thr308 and Ser473, that are normally induced on PKB following stimulation of the cells with insulin or insulin-like growth factor-1 (IGF-1). A dominant negative form of phosphoinositide 3-kinase (PI3-K) did not affect m/p-PKBalpha activity. The pleckstrin homology (PH) domain of m/p-PKBalpha was not required for its activation or phosphorylation on Thr308 and Ser473, suggesting that this domain may serve as a membrane-targeting module. Consistent with this view, PKBalpha was translocated to the plasma membrane within minutes after stimulation with IGF-1. This translocation required the PH domain and was sensitive to wortmannin. Our results indicate that PI3-K activity is required for translocation of PKB to the plasma membrane, where its activation occurs through phosphorylation of the same sites that are induced by insulin or IGF-1. Following activation the kinase detached from the membrane and translocated to the nucleus.

Mechanism of activation of protein kinase B by insulin and IGF-1.

Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).

The variable subunit associated with protein phosphatase 2A0 defines a novel multimember family of regulatory subunits.

Two protein phosphatase 2A (PP2A) holoenzymes were isolated from rabbit skeletal muscle containing, in addition to the catalytic and PR65 regulatory subunits, proteins of apparent molecular masses of 61 and 56 kDa respectively. Both holoenzymes displayed low basal phosphorylase phosphatase activity, which could be stimulated by protamine to an extent similar to that of previously characterized PP2A holoenzymes. Protein micro-sequencing of tryptic peptides derived from the 61 kDa protein, termed PR61, yielded 117 residues of amino acid sequence. Molecular cloning by enrichment of specific mRNAs, followed by reverse transcription-PCR and cDNA library screening, revealed that this protein exists in multiple isoforms encoded by at least three genes, one of which gives rise to several splicing variants. Comparisons of these sequences with the available databases identified one more human gene and predicted another based on a rabbit cDNA-derived sequence, thus bringing the number of genes encoding PR61 family members to five. Peptide sequences derived from PR61 corresponded to the deduced amino acid sequences of either alpha or beta isoforms, indicating that the purified PP2A preparation was a mixture of at least two trimers. In contrast, the 56 kDa subunit (termed PR56) seems to correspond to the epsilon isoform of PR61. Several regulatory subunits of PP2A belonging to the PR61 family contain consensus sequences for nuclear localization and might therefore target PP2A to nuclear substrates.

Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors.

Treatment of quiescent Swiss 3T3 fibroblasts with serum, or with the phosphatase inhibitors okadaic acid and vanadate, induced a 2- to 11-fold activation of the serine/ threonine RAC protein kinase (RAC-PK). Kinase activation was accompanied by decreased mobility of RAC-PK on SDS/PAGE such that three electrophoretic species (a to c) of the kinase were detected by immunoblot analysis, indicative of differentially phosphorylated forms. Addition of vanadate to arrested cells increased the RAC-PK phosphorylation level 3-to 4-fold. Unstimulated RAC-PK was phosphorylated predominantly on serine, whereas the activated kinase was phosphorylated on both serine and threonine residues. Treatment of RAC-PK in vitro with protein phosphatase 2A led to kinase inactivation and an increase in electrophoretic mobility. Deletion of the N-terminal region containing the pleckstrin homology domain did not affect RAC-PK activation by okadaic acid, but it reduced vanadate-stimulated activity and also blocked the serum-induced activation. Deletion of the serine/threonine rich C-terminal region impaired both RAC-PKalpha basal and vanadate-stimulated activity. Studies using a kinase-deficient mutant indicated that autophosphorylation is not involved in RAC-PKalpha activation. Stimulation of RAC-PK activity and electrophoretic mobility changes induced by serum were sensitive to wortmannin. Taken together the results suggest that RAC-PK is a component of a signaling pathway regulated by phosphatidylinositol (PI) 3-kinase, whose action is required for RAC-PK activation by phosphorylation.

Molecular cloning and characterization of a conserved nuclear serine(threonine) protein kinase.

Human, Drosophila melanogaster, and Caenorhabditis elegans cDNA clones encoding homologues of a serine(threonine) protein kinase (EC 2.7.1.37) (designated Ndr protein kinase) have been isolated and sequenced. The human and Drosophila cDNAs predict polypeptides of 54 kDa and 52 kDa, respectively, which share approximately 80% amino acid similarity. Northern analysis of human tissues revealed a ubiquitously expressed 3.9-kb transcript. Recombinant GST-Ndr underwent intramolecular autophosphorylation on serine and threonine residues in vitro but failed to transphosphorylate several standard protein kinase substrates. Transfection of the human cDNA into COS-1 cells resulted in the appearance of an intense nuclear staining in cells analyzed by indirect immunofluorescence; deletion mutagenesis identified a short basic peptide, KRKAETWKRNRR, responsible for the nuclear accumulation of Ndr. Thus, Ndr is a conserved and widely expressed nuclear protein kinase. The closest known relative of this previously uncharacterized kinase is Dbf2, a budding yeast protein kinase required for the completion of nuclear division.

Developmental regulation of expression and activity of multiple forms of the Drosophila RAC protein kinase.

We have characterized the Drosophila homologue of the proto-oncogenic RAC protein kinase (DRAC-PK). The DRAC-PK gene gives rise to two transcripts with the same coding potential, generated by the use of two different polyadenylation signals. Each transcript encodes two polypeptides because of the presence of a weaker initiator ACG codon, upstream from the major AUG, such that the larger protein contains an N-terminal extension. Like the human isoforms, DRAC-PKs possess a novel signaling region, the pleckstrin homology domain. DRAC-PK proteins have a similar expression pattern, being regulated both maternally and zygotically, and are expressed throughout Drosophila development. Antisera specific for recombinant DRAC-PK and for its C terminus detected two polypeptides of 66 and 85 kDa in Drosophila extracts. The antirecombinant antisera also recognized a polypeptide of 120 kDa from Drosophila, which apparently shared an epitope related to DRAC-PK sequences. The role of p120 appears to be restricted compared with that of DRAC-PK, since it was not detected in larvae or adult flies. There was no spatial restriction of DRAC-PK expression during embryogenesis, suggesting that localized activation might be a regulatory mechanism for its function. DRAC-PK possesses an intrinsic kinase activity that is approximately 8-fold higher in adult flies than in 0-3-h embryos undergoing rapid mitotic cycles.

Isolation, characterization, and chromosomal localization of the porcine calcitonin receptor gene. Identification of two variants of the receptor generated by alternative splicing.

The gene encoding the calcitonin receptor (CTR) was isolated from a porcine kidney epithelial cell line (LLC-PK1) genomic library and found to span approximately 70 kilobases. Analysis of the gene sequence revealed that the CTR mRNA encompasses 14 exons with 12 exons encoding the protein. Two splicing acceptor sites separated by 48 nucleotides were found in intron 7. The expression of two mRNA species in LLC-PK1 cells was subsequently confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequencing. In LLC-PK1 cells the mRNA encoding the shorter CTR (CTR-1a) is approximately 1,000 times more abundant than the longer variant (CTR-1b), as estimated by the competitive RT-PCR. The transcription initiation site of the CTR gene was mapped by primer extension, S1 nuclease, and RT-PCR analysis. The proximal promoter region of 500 base pair is GC-rich (66%) and CpG-rich (CpG/GpC ratio 0.71). Transient transfection of CTR gene promoter-luciferase chimeras in LLC-PK1 cells led to the expression of luciferase activity. The CTR gene was mapped to chromosome band 9q11-q12.

Structure and expression of a 72-kDa regulatory subunit of protein phosphatase 2A. Evidence for different size forms produced by alternative splicing.

The trimeric form of protein phosphatase 2A consisting of 36-, 65-, and 72-kDa subunits (previously termed polycation-stimulated protein phosphatase M) was purified from rabbit skeletal muscle. Amino acid sequence data of the 72-kDa regulatory subunit (termed PR72) were used to isolate cDNAs from human heart and fetal brain libraries and libraries derived from WI-38 and MCF-7 cells. The clones isolated from the heart cDNA library revealed an open reading frame encoding a protein with a predicted molecular mass of 62 kDa. All the peptides sequenced from the protein matched with the sequence predicted from the cDNA. However, in vitro transcription and translation from this cDNA yielded a protein with an apparent molecular mass of 72 kDa on sodium dodecyl sulfate-polyacrylamide gels. From brain we isolated cDNA clones spanning an open reading frame encoding a 130-kDa protein (termed PR130). The apparent molecular mass of the protein produced by in vitro transcription and translation was 130 kDa. This protein has exactly the same deduced C-terminal protein sequence as the PR72 subunit from amino acids 45 to 527 but has an N-terminal extension of 665 amino acids. It is likely, therefore, that these two proteins arise from the same gene by alternative splicing. In human tissues several transcripts were detected by Northern analysis generated probably by the use of different polyadenylation signals and alternative splicing. High levels of the PR72 mRNAs were detected in heart and muscle, while lower levels of PR130 transcripts were found in heart, brain, placenta, lung, muscle, and kidney.

Structure of the 55-kDa regulatory subunit of protein phosphatase 2A: evidence for a neuronal-specific isoform.

The trimeric form of protein phosphatase 2A (PP2A1 or polycation-stimulated protein phosphatase H1) was purified to homogeneity from rabbit skeletal muscle. Preparative SDS-polyacrylamide gel electrophoresis was used to purify the individual subunits with relative molecular masses of 36, 55, and 65 kDa. Sequence analysis of five peptides from the 65-kDa regulatory subunit (PR65) suggested that it was identical with the PR65 subunit derived from the dimeric protein phosphatase 2A2. Amino acid sequences derived from the 55-kDa regulatory subunit (PR55) were used to clone human and rabbit cDNAs encoding this protein. The PR55 subunit was found to be encoded by two genes, termed alpha and beta. The open reading frames of the PR55 alpha and beta cDNAs spanned 1341 and 1329 nucleotides, respectively, and predicted proteins with a molecular mass of about 52 kDa that are 86% identical. Comparison of the human PR55 amino acid sequences with the data obtained from the rabbit skeletal muscle protein and a partial rabbit PR55 beta cDNA clone indicated a high degree of conservation. Analysis of the mRNA expression in human cell lines revealed that the PR55 alpha isoform was encoded by two transcripts of about 2.3 and 2.5 kb and a less abundant 4.4-kb mRNA. Whereas a PR55 beta transcript of about 2.3 kb was detected at high levels in the neuroblastoma derived cell line LA-N-1, the level of the mRNA was very low in the other human cell lines analyzed. Interestingly, the PR55 sequence showed limited homology to the catalytic domain (domains VI-IX) of the c-abl protein tyrosine kinase.