6-mer peptide selectively anneals to a pathogenic serpin conformation and blocks polymerization. Implications for the prevention of Z alpha(1)-antitrypsin-related cirrhosis.

Conformational diseases such as amyloidosis, Alzheimer's disease, prion diseases, and the serpinopathies are all caused by structural rearrangements within a protein that transform it into a pathological species. These diseases are typified by the Z variant of alpha(1)-antitrypsin (E342K), which causes the retention of protein within hepatocytes as inclusion bodies that are associated with neonatal hepatitis and cirrhosis. The inclusion bodies result from the Z mutation perturbing the conformation of the protein, which facilitates a sequential interaction between the reactive center loop of one molecule and beta-sheet A of a second. Therapies to prevent liver disease must block this reactive loop-beta-sheet polymerization without interfering with other proteins of similar tertiary structure. We have used reactive loop peptides to explore the differences between the pathogenic Z and normal M alpha(1)-antitrypsin. The results show that the reactive loop is likely to be partially inserted into beta-sheet A in Z alpha(1)-antitrypsin. This conformational difference from M alpha(1)-antitrypsin was exploited with a 6-mer reactive loop peptide (FLEAIG) that selectively and stably bound Z alpha(1)-antitrypsin. The importance of this finding is that the peptide prevented the polymerization of Z alpha(1)-antitrypsin and did not significantly anneal to other proteins (such as antithrombin, alpha(1)-antichymotrypsin, and plasminogen activator inhibitor-1) with a similar tertiary structure. These findings provide a lead compound for the development of small molecule inhibitors that can be used to treat patients with Z alpha(1)-antitrypsin deficiency. Furthermore they demonstrate how a conformational disease process can be selectively inhibited with a small peptide.     

Disruption of the 290-342 salt bridge is not responsible for the secretory defect of the PiZ alpha 1-antitrypsin variant

Crystallographic studies have previously suggested that Lys290 forms a salt bridge with Glu342 in the serine protease inhibitor alpha 1-antitrypsin. Disruption of the formation of this structural feature by a Glu to Lys substitution at residue 342 in the PiZ variant has been implicated in causing the defective secretion of this mutant protein from hepatocytes (10-15% of normal). To test the validity of this hypothesis, mutant human alpha 1-antitrypsin cDNA constructs coding for specific amino acid substitutions at residues 290 and 342 were generated and the corresponding mutant proteins were expressed in mouse hepatoma cells. When the potential to form the salt bridge was reestablished by a Lys290 to Glu290 substitution in the PiZ variant, its secretion was increased to only 38% of normal. Furthermore, disruption of this structural feature by a Lys290 to Glu290 substitution in the normal inhibitor failed to reduce the secretion of alpha 1-antitrypsin to the extent observed for the PiZ variant (73% of normal). Finally, substitution of the neutral amino acid Gln at residue 342 only reduced the secretion of alpha 1-antitrypsin to 55% of normal. Of all mutant proteins tested, those bearing Lys at position 342 were secreted at the lowest levels. These findings demonstrate that although disruption of the 290-342 salt bridge does affect the secretion of alpha 1-antitrypsin, it is the substitution of Lys at residue 342 that causes the dramatic secretory defect of the PiZ variant.     

Alpha(1)-antitrypsin deficiency,liver disease and emphysema

alpha(1)-Antitrypsin is a member of the serine proteinase inhibitor (serpin) superfamily and a potent inhibitor of neutrophil elastase. The most important deficiency variant of alpha(1)-antitrypsin arises from the Z mutation (Glu342Lys). This mutation perturbs the protein's tertiary structure to promote a precise, sequential intermolecular linkage that results in polymer formation. These polymers accumulate within the endoplasmic reticulum of the hepatocyte forming inclusion bodies that are associated with neonatal hepatitis, juvenile cirrhosis and adult hepatocellular carcinoma. The resultant secretory defect leads to plasma deficiency of alpha(1)-antitrypsin. This exposes lung tissue to uncontrolled proteolytic attack from neutrophil elastase, culminating in alveolar destruction. Thus, the Z alpha(1)-antitrypsin homozygote is predisposed to developing early onset basal, panacinar emphysema. In this review, we summarise the current understanding of the pathobiology of alpha(1)-antitrypsin deficiency and the associated liver cirrhosis and emphysema. We show how this knowledge has led to the development of novel therapeutic approaches to treat this condition.     

Identification of a second mutation in the protein-coding sequence of the Z type alpha 1-antitrypsin gene

This study reports the entire nucleotide sequence of the protein coding region sequence of the alpha 1-antitrypsin (alpha 1AT) Z gene, a common form of the alpha 1AT gene associated with serum alpha 1AT deficiency. In addition to Glu342 to Lys342 mutation in exon V which has been previously identified by peptide analysis, another point mutation (GTG to GCG in exon III) in the gene sequence predicts a second amino acid substitution (Val213 to Ala213) in the Z protein. This Val213 to Ala213 mutation was confirmed to be a general finding in Z type alpha 1AT gene by evaluating genomic DNA from 40 Z haplotypes using synthetic oligonucleotide gene probes directed toward the mutated exon III sequences in the Z gene. Furthermore, the exon III Val213 to Ala213 mutation eliminates a BstEII restriction endonuclease site in the alpha 1AT Z gene, allowing rapid identification of this Val213 to Ala213 substitution at the genomic DNA level. Surprisingly, when genomic DNA samples from individuals thought to be homozygous for the M1 gene (the most common alpha 1AT normal haplotype) were evaluated with BstEII, 23% of the M1 haplotypes were BstEII site negative, thus identifying a new form of M1 (i.e. M1(Ala213], likely identical to M1 but with an isoelectric focusing "silent" amino acid substitution (Val213 to Ala213). Although the relative importance of the newly identified exon III Val213 to Ala213 mutation to the pathogenesis of the abnormalities associated with the Z gene is not known, it is likely that M1(Ala213) gene represents a common "normal" polymorphism of the alpha 1AT gene that served as an evolutionary intermediate between the M1(Val213) and Z genes.     

Insertion of an elastase-binding loop into interleukin-1 beta

The protease-binding sequence EAIPMSIPPE from alpha 1-antitrypsin has been inserted into the cytokine interleukin-1 beta, replacing residues 50-53. The resulting mutant protein was cleaved specifically at a single site by elastase and chymotrypsin, but not by trypsin. The cleavage by elastase was shown to be between Met and Ser of the inserted loop. In contrast, wild-type interleukin is not susceptible to cleavage by any of these enzymes. The mutant protein acts as an inhibitor of elastase, with a KI of approximately 30 microM. The wild type displays no such inhibitory activity. The overall structure of the mutant, as demonstrated by CD, appears to be indistinguishable from that of the wild type. These results indicate that the protease-binding region of alpha 1-antitrypsin can be recognized and is active even within the context of an entirely different protein structure. Given that interleukin-1 beta binds to, and is internalized by, many types of cells, this hybrid protein also demonstrates the feasibility of using interleukin-1 beta as a delivery system for useful therapeutic agents.     

A kinetic mechanism for the polymerization of alpha1-antitrypsin

The mutation in the Z deficiency variant of alpha1-antitrypsin perturbs the structure of the protein to allow a unique intermolecular linkage. These loop-sheet polymers are retained within the endoplasmic reticulum of hepatocytes to form inclusions that are associated with neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. The process of polymer formation has been investigated here by intrinsic tryptophan fluorescence, fluorescence polarization, circular dichroic spectra and extrinsic fluorescence with 8-anilino-1-naphthalenesulfonic acid and tetramethylrhodamine-5-iodoacetamide. These biophysical techniques have demonstrated that alpha1-antitrypsin polymerization is a two-stage process and have allowed the calculation of rates for both of these steps. The initial fast phase is unimolecular and likely to represent temperature-induced protein unfolding, while the slow phase is bimolecular and associated with loop-sheet interaction and polymer formation. The naturally occurring Z, S, and I variants and recombinant site-directed reactive loop and shutter domain mutants of alpha1-antitrypsin were used to demonstrate the close association between protein stability and rate of alpha1-antitrypsin polymerization. Taken together, these data allow us to propose a kinetic mechanism for alpha1-antitrypsin polymer formation that involves the generation of an unstable intermediate, which can form polymers or generate latent protein.     

Alpha 1-antitrypsin deficiency—A defect in secretion

A naturally occurring point mutation in the human alpha 1-antitrypsin gene leads to the synthesis of a variant of the protein which is poorly secreted from hepatocytes. This Z; mutation codes for a glutamic acid to lysine substitution at residue 342 in the polypeptide chain. The mutant protein is correctly translocated into the lumen of the endoplasmic reticulum and core glycosylated but inefficiently transported beyond the ER compartment. Experiments using Xenopus oocytes as a surrogate secretory cell show that abberant secretion of the variant is not confined to hepatocytes and glycosylation of the polypeptide is not obligatory for the block in secretion. Site-directed mutagenesis can be used to examine the effect of natural mutations on protein structure and the relationship between structure and intraceltular transport.     

Aggregation of plasma Z type alpha 1-antitrypsin suggests basic defect for the deficiency

The abnormal type of alpha 1-antitrypsin, PI (protease inhibitor) type Z, is associated with inclusion bodies in the liver, which contain non-secreted alpha 1-antitrypsin. Our studies show that Z protein has an inherent tendency to aggregate, even in plasma. Depending upon conditions, from 15 to 70% of the Z protein in plasma was in a high-Mr form, compared with 1.5% of M type alpha 1-antitrypsin. The high-Mr complex in plasma cannot be disaggregated using Triton X detergent or reducing conditions. This increased tendency to aggregate can be explained by the mutation affecting, tertiary structure and salt bridge formation in Z protein. We have observed this same tendency to aggregate for Mmalton alpha 1-antitrypsin, a rarer variant also associated with a plasma deficiency.     

Crystallographic and Cellular Characterisation of Two Mechanisms Stabilising the Native Fold of α1-Antitrypsin: Implications for Disease and Drug Design

The common Z mutant (Glu342Lys) of α₁-antitrypsin results in the formation of polymers that are retained within hepatocytes. This causes liver disease whilst the plasma deficiency of an important proteinase inhibitor predisposes to emphysema. The Thr114Phe and Gly117Phe mutations border a surface cavity identified as a target for rational drug design. These mutations preserve inhibitory activity but reduce the polymerisation of wild-type native α₁-antitrypsin in vitro and increase secretion in a Xenopus oocyte model of disease. To understand these effects, we have crystallised both mutants and solved their structures. The 2.2 Å structure of Thr114Phe α₁-antitrypsin demonstrates that the effects of the mutation are mediated entirely by well-defined partial cavity blockade and allows in silico screening of fragments capable of mimicking the effects of the mutation. The Gly117Phe mutation operates differently, repacking aromatic side chains in the helix F-β-sheet A interface to induce a half-turn downward shift of the adjacent F helix. We have further characterised the effects of these two mutations in combination with the Z mutation in a eukaryotic cell model of disease. Both mutations increase the secretion of Z α₁-antitrypsin in the native conformation, but the double mutants remain more polymerogenic than the wild-type (M) protein. Taken together, these data support different mechanisms by which the Thr114Phe and Gly117Phe mutations stabilise the native fold of α₁-antitrypsin and increase secretion of monomeric protein in cell models of disease.     

The Z Mutation Alters the Global Structural Dynamics of α1-Antitrypsin

α₁-Antitrypsin (α₁AT) deficiency, the most common serpinopathy, results in both emphysema and liver disease. Over 90% of all clinical cases of α₁AT deficiency are caused by the Z variant in which Glu342, located at the top of s5A, is replaced by a Lys which results in polymerization both in vivo and in vitro. The Glu342Lys mutation removes a salt bridge and a hydrogen bond but does not effect the thermodynamic stability of Z α₁AT compared to the wild type protein, M α₁AT, and so it is unclear why Z α₁AT has an increased polymerization propensity. We speculated that the loss of these interactions would make the native state of Z α₁AT more dynamic than M α₁AT and that this change renders the protein more polymerization prone. We have used hydrogen/deuterium exchange combined with mass spectrometry (HXMS) to determine the structural and dynamic differences between native Z and M α₁AT to reveal the molecular basis of Z α₁AT polymerization. Our HXMS data shows that the Z mutation significantly perturbs the region around the site of mutation. Strikingly the Z mutation also alters the dynamics of regions distant to the mutation such as the B, D and I helices and specific regions of each β-sheet. These changes in global dynamics may lead to an increase in the likelihood of Z α₁AT sampling a polymerogenic structure thereby causing disease.     

All-Atom Simulations Reveal How Single-Point Mutations Promote Serpin Misfolding

Protein misfolding is implicated in many diseases, including serpinopathies. For the canonical inhibitory serpin α₁-antitrypsin, mutations can result in protein deficiencies leading to lung disease, and misfolded mutants can accumulate in hepatocytes, leading to liver disease. Using all-atom simulations based on the recently developed bias functional algorithm, we elucidate how wild-type α₁-antitrypsin folds and how the disease-associated S (Glu264Val) and Z (Glu342Lys) mutations lead to misfolding. The deleterious Z mutation disrupts folding at an early stage, whereas the relatively benign S mutant shows late-stage minor misfolding. A number of suppressor mutations ameliorate the effects of the Z mutation, and simulations on these mutants help to elucidate the relative roles of steric clashes and electrostatic interactions in Z misfolding. These results demonstrate a striking correlation between atomistic events and disease severity and shine light on the mechanisms driving chains away from their correct folding routes.     

Targeting a surface cavity of alpha 1-antitrypsin to prevent conformational disease

Conformational diseases are caused by a structural rearrangement within a protein that results in aberrant intermolecular linkage and tissue deposition. This is typified by the polymers that form with the Z deficiency variant of alpha 1-antitrypsin (Glu-342 --> Lys). These polymers are retained within hepatocytes to form inclusions that are associated with hepatitis, cirrhosis, and hepatocellular carcinoma. We have assessed a surface hydrophobic cavity in alpha1-antitrypsin as a potential target for rational drug design in order to prevent polymer formation and the associated liver disease. The introduction of either Thr-114 --> Phe or Gly-117 --> Phe on strand 2 of beta-sheet A within this cavity significantly raised the melting temperature and retarded polymer formation. Conversely, Leu-100 --> Phe on helix D accelerated polymer formation, but this effect was abrogated by the addition of Thr-114 --> Phe. None of these mutations affected the inhibitory activity of alpha 1-antitrypsin. The importance of these observations was underscored by the finding that the Thr-114 --> Phe mutation reduced polymer formation and increased the secretion of Z alpha 1-antitrypsin from a Xenopus oocyte expression system. Moreover cysteine mutants within the hydrophobic pocket were able to bind a range of fluorophores illustrating the accessibility of the cavity to external agents. These results demonstrate the importance of this cavity as a site for drug design to ameliorate polymerization and prevent the associated conformational disease.     

Hypersensitive mousetraps,alpha1-antitrypsin deficiency and dementia

Alpha(1)-antitrypsin functions as a "mousetrap" to inhibit its target proteinase, neutrophil elastase. The common severe Z deficiency variant (Glu(342)-->Lys) destabilizes the mousetrap to allow a sequential protein-protein interaction between the reactive-centre loop of one molecule and beta-sheet A of another. These loop-sheet polymers accumulate within hepatocytes to form inclusion bodies that are associated with juvenile cirrhosis and hepatocellular carcinoma. The lack of circulating protein predisposes the Z alpha(1)-antitrypsin homozygote to emphysema. Loop-sheet polymerization is now recognized to underlie deficiency variants of other members of the serine proteinase inhibitor (serpin) superfamily, i.e. antithrombin, C1 esterase inhibitor and alpha(1)-antichymotrypsin, which are associated with thrombosis, angio-oedema and emphysema respectively. Moreover, we have shown recently that the same process in a neuron-specific protein, neuroserpin, underlies a novel inclusion-body dementia, known as familial encephalopathy with neuroserpin inclusion bodies. Our understanding of the structural basis of polymerization has allowed the development of strategies to prevent the aberrant protein-protein interaction in vitro. This must now be achieved in vivo if we are to treat the associated clinical syndromes.     

Structural change in β-sheet A of Z α(1)-antitrypsin is responsible for accelerated polymerization and disease

The presence of the Z mutation (Glu342Lys) is responsible for more than 95% of α₁-antitrypsin (α₁AT) deficiency cases. It leads to increased polymerization of the serpin α₁AT during its synthesis and in circulation. It has been proposed that the Z mutation results in a conformational change within the folded state of antitrypsin that enhances its polymerization. In order to localize the conformational change, we have created two single tryptophan mutants of Z α₁AT and analyzed their fluorescence properties. α₁AT contains two tryptophan residues that are located in distinct regions of the molecule: Trp194 at the top of β-sheet A and Trp238 on β-sheet B. We have replaced each tryptophan residue individually with a phenylalanine in order to study the local environment of the remaining tryptophan residue in both M and Z α₁AT. A detailed fluorescence spectroscopic analysis of each mutant was carried out, and we detected differences in the emission spectrum, the Stern-Volmer constant for potassium iodide quenching and the anisotropy of only Trp194 in Z α₁AT compared to M α₁AT. Our data reveal that the Z mutation results in a conformational change at the top of β-sheet A but does not affect the structural integrity of β-sheet B.     

Sequence data of the rare deficient alpha 1-antitrypsin variant PI Zaugsburg.

Weidinger et al. recognized a rare deficient PI-variant, named PI Zaugsburg (PI Zaug), by using isoelectric focusing with a narrow pH gradient. The serum alpha 1-antitrypsin (alpha 1AT) level determined quantitatively in an individual carrying the phenotype PI M1Zaug revealed a value of 50%-60% of the normal range. The frequency of the deficient PI*Zaug allele is still unknown. Haplotyping the Zaug-affected chromosome, we found a pattern different from the common PI*Z allele described by Cox et al. Therefore, we directly sequenced the coding exons of both genes (M1 and Zaug) after PCR amplification. Zaug sequence data analysis showed the presence of the common PI*Z allele-specific mutation (M1 Glu342 GAG to Z Lys342 AAG) surprisingly occurring in an M2 ancestral gene. This is not consistent with the heretofore common finding, by Nukiwa et al. and others, that this mutation is derived from an M1 (Ala213) background gene. No further mutations were found in the PI Zaug gene.     

Construction and expression of alpha 1-proteinase inhibitor mutants and the effects of these mutations on secretion of the variant inhibitors

Human alpha 1-proteinase inhibitor (A1Pi) deficiency, associated with the Z variant A1Pi gene, results from defective secretion of the inhibitor from the liver and appears to be a direct consequence of replacement of Glu342 with Lys. To investigate the effect of the amino acid occupying position 342 on secretion of A1Pi, we have used oligonucleotide-directed mutagenesis of A1Pi cDNA to randomly change the codon specifying this amino acid. Since replacement of Glu342 by Lys leads to a change in the predicted secondary structure for this protein, we also tested the possibility that defective secretion of A1PiZ is the result of this type of alteration. For this purpose, site-directed mutagenesis was used to produce sequences encoding A1Pi retaining Glu342 but predicted to have A1PiZ type secondary structure. The effects of 10 different amino acids occupying position 342 on the secretion of A1Pi were determined by pulse-chase experiments and by enzyme-linked immunosorbent assay of medium from transiently transfected COS cells. Results of these studies show that secretion of A1Pi is most efficient when position 342 is occupied by a negatively charged amino acid, efficient but somewhat less so when occupied by a neutral amino acid, and least efficient when a positively charged residue is present. The mutation designed to alter secondary structure had no effect on the secretion of A1Pi. As indicated by immunofluorescence microscopy and mobility of intracellular A1Pi on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lowered secretion is accompanied by accumulation of A1Pi in the endoplasmic reticulum of the transfected cells. These results are compatible with the ideas that secretion of A1Pi is directly influenced by the amino acid occupying position 342, that a positively charged amino acid in this position is especially detrimental to secretion of this protein, and that the rate-limiting step in the secretion of the altered forms is transport from endoplasmic reticulum to Golgi.     

The effect of amino acid substitutions at position 342 on the secretion of human alpha 1-antitrypsin from Xenopus oocytes

A glutamic acid to lysine change in the Z variant of human alpha 1-antitrypsin is associated with a failure to secrete the protein from synthesising cells. The block in export of the protein may be caused either by the loss of an acidic residue or the introduction of a basic one at this point in the polypeptide chain. Site-directed mutagenesis has been used to construct novel alpha 1-antitrypsin mutants which show that the side chain interactions from Glu-342 are not obligatory for protein export and it is rather the introduction of a basic residue at this point which produces the intracellular accumulation of the protein.     

Sugar and alcohol molecules provide a therapeutic strategy for the serpinopathies that cause dementia and cirrhosis

Mutations in neuroserpin and alpha1-antitrypsin cause these proteins to form ordered polymers that are retained within the endoplasmic reticulum of neurones and hepatocytes, respectively. The resulting inclusions underlie the dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) and Z alpha1-antitrypsin-associated cirrhosis. Polymers form by a sequential linkage between the reactive centre loop of one molecule and beta-sheet A of another, and strategies that block polymer formation are likely to be successful in treating the associated disease. We show here that glycerol, the sugar alcohol erythritol, the disaccharide trehalose and its breakdown product glucose reduce the rate of polymerization of wild-type neuroserpin and the Ser49Pro mutant that causes dementia. They also attenuate the polymerization of the Z variant of alpha1-antitrypsin. The effect on polymerization was apparent even when these agents had been removed from the buffer. None of these agents had any detectable effect on the structure or inhibitory activity of neuroserpin or alpha1-antitrypsin. These data demonstrate that sugar and alcohol molecules can reduce the polymerization of serpin mutants that cause disease, possibly by binding to and stabilizing beta-sheet A.     

Regulator of G Signaling 16 is a marker for the distinct endoplasmic reticulum stress state associated with aggregated mutant alpha1-antitrypsin Z in the classical form of alpha1-antitrypsin deficiency

In the classical form of alpha(1)-antitrypsin deficiency, a mutant protein accumulates in a polymerized form in the endoplasmic reticulum (ER) of liver cells causing liver damage and carcinogenesis by a gain-of-toxic function mechanism. Recent studies have indicated that the accumulation of mutant alpha(1)-antitrypsin Z in the ER specifically activates the autophagic response but not the unfolded protein response and that autophagy plays a critical role in disposal of insoluble alpha(1)-antitrypsin Z. In this study, we used genomic analysis of the liver in a novel transgenic mouse model with inducible expression to screen for changes in gene expression that would potentially define how the liver responds to accumulation of this mutant protein. There was no unfolded protein response. Of several distinct gene expression profiles, marked up-regulation of regulator of G signaling (RGS16) was particularly notable. RGS16 did not increase when model systems were exposed to classical inducers of ER stress, including tunicamycin and calcium ionophore, or when a nonpolymerogenic alpha(1)-antitrypsin mutant accumulated in the ER. RGS16 was up-regulated in livers from patients with alpha(1)-antitrypsin deficiency, and the degree of up-regulation correlated with the hepatic levels of insoluble alpha(1)-antitrypsin Z protein. Taken together, these results indicate that expression of RGS16 is an excellent marker for the distinct form of "ER stress" that occurs in alpha(1)-antitrypsin deficiency, presumably determined by the aggregation-prone properties of the mutant protein that characterizes the deficiency.     

Consequences of lysine 72 mutation on the phosphorylation and activation state of cAMP-dependent kinase

General strategies to obtain inactive kinases have utilized mutation of key conserved residues in the kinase core, and the equivalent Lys72 in cAMP-dependent kinase has often been used to generate a "dead" kinase. Here, we have analyzed the consequences of this mutation on kinase structure and function. Mutation of Lys72 to histidine (K72H) generated an inactive enzyme, which was unphosphorylated. Treatment with an exogenous kinase (PDK-1) resulted in a mutant that was phosphorylated only at Thr197 and remained inactive but nevertheless capable of binding ATP. Ser338 in K72H cannot be autophosphorylated, nor can it be phosphorylated in an intermolecular process by active wild type C-subunit. The Lys72 mutant, once phosphorylated on Thr197, can bind with high affinity to the RIalpha subunits. Thus a dead kinase can still act as a scaffold for binding substrates and inhibitors; it is only phosphoryl transfer that is defective. Using a potent inhibitor of C-subunit activity, H-89, Escherichia coli-expressed C-subunit was also obtained in its unphosphorylated state. This protein is able to mature into its active form in the presence of PDK-1 and is able to undergo secondary autophosphorylation on Ser338. Unlike the H-89-treated wild type protein, the mutant protein (K72H) cannot undergo the subsequent cis autophosphorylation following phosphorylation at Thr197. Using these two substrates and mammalian-expressed PDK-1, we can elucidate a possible two-step process for the activation of the C-subunit: initial phosphorylation on the activation loop at Thr197 by PDK-1, or a PDK-1-like enzyme, followed by second cis autophosphorylation step at Ser338.