Definition of the inhibitory domain of smooth muscle myosin light chain kinase by site-directed mutagenesis

Site-directed mutagenesis of smooth muscle myosin light chain kinase was applied to define its autoinhibitory domain. Mutants were all initiated at Leu-447 but contained varying lengths of C-terminal sequence. Those containing the complete C-terminal sequence to Glu-972 possessed kinase activities that were calmodulin-dependent. Removal of the putative inhibitory domain by truncation to Thr-778 resulted in generation of a constitutively active (calmodulin-independent) species. Thus, the inhibitory domain lies to the C-terminal side of Thr-778. Truncation to Lys-793 and to Trp-800 also resulted in constitutively active mutants, although the specific activity of the latter was less than the other mutants. None of the truncated mutants bound calmodulin. For each mutant, the Km values with respect to ATP and to the 20,000-dalton light chain were similar to values obtained with the native enzyme. The presence of the inhibitory domain was detected by activation of kinase activity following limited proteolysis with trypsin. Using this procedure, it was determined that the inhibitory domain was manifest only in the mutant truncated to Trp-800 and was absent from that ending at Lys-793. These results indicate that a critical region of the inhibitory domain is contained within the sequence Tyr-794 to Trp-800. This region overlaps with the calmodulin-binding site for five residues. Our assignment of the inhibitory sequence is consistent with autoinhibition via a pseudosubstrate domain.     

Primary structure required for the inhibition of smooth muscle myosin light chain kinase.

Myosin light chain kinase (MLCK) contains the autoinhibitor sequence right next to the N-terminus side of the calmodulin binding region. In this paper, the structural requirement of the inhibition of MLCK activity was studied using synthetic peptide analogs. Peptides Ala-783-Lys-799 and Ala-783-Arg-798 inhibited calmodulin independent MLCK at the same potency as the peptide Ala-783-Gly-804. Deletion of Arg-797-Lys-799 or substitution of these residues to Ala markedly increased the Ki while the substitution of Lys-792 and Lys-793 to Ala and the deletion of Lys-784-Lys-785 did not affect the inhibitory activity of the peptides. The results suggest that Arg-797-Arg-798 are especially important for the inhibitory activity among other basic residues in the autoinhibitory region.     

Proteolytic cleavage sites in smooth muscle myosin-light-chain kinase and their relation to structural and regulatory domains

Proteolysis of the smooth muscle myosin-light-chain kinase with either thermolysin or endoproteinase Lys-C cleaves the enzyme towards the amino-terminus between the first and second unc domains, unc-II-1 and unc-II-2, and in the calmodulin-binding domain. The thermolytic fragment extends 532 residues from Ser275 to Ala806 and is resistant to further digestion. It is catalytically inactive and does not bind calmodulin. Further proteolysis of the thermolytic fragment with trypsin generates a constitutively active fragment. Digestion with endoproteinase Lys-C initially results in an inactive fragment of 516 residues, Ala287 to Lys802. Further digestion with Lys-C endoproteinase results in a constitutively active 474-residue fragment with the same amino-terminus, but a carboxyl-terminus at Lys760, near Arg762, the last conserved residue of protein kinase catalytic domains. There is no cleavage in the acidic-residue-rich connecting peptide between the amino-terminus of the catalytic domain and the unc-I domain, nor within the unc-II or unc-I domains or between the adjacent unc-II-2 and unc-I domains. The pattern of cleavages by these proteases reflects well the predicted domain structure of the myosin-light-chain kinase and further delineates the regulatory pseudosubstrate region. A synthetic peptide corresponding to the pseudosubstrate sequence, MLCK(787-807) was a more potent inhibitor by three orders of magnitude than the overlapping peptide MLCK(777-793) proposed by Ikebe et al. (1989) [Ikebe, M., Maruta, S. & Reardon, S. (1989) J. Biol. Chem. 264, 6967-6971] to be important in autoregulation of the myosin-light-chain kinase.     

Autoinhibition and isoform-specific dominant negative inhibition of the type II cGMP-dependent protein kinase

In the absence of cyclic nucleotides, the cAMP-dependent protein kinase and cGMP-dependent protein kinases (cGKs) suppress phosphotransfer activity at the catalytic cleft by competitive inhibition of substrate binding with a pseudosubstrate sequence within the holoenzyme. The magnitude of inhibition can be diminished by autophosphorylation near this pseudosubstrate sequence. Activation of type I cGK (cGKI) and type II cGK (cGKII) are differentially regulated by their cyclic nucleotide-binding sites. To address the possibility that the distinct activation mechanisms of cGKII and cGKI result from differences in the autophosphorylation of the inhibitory domain, we investigated the effects of autophosphorylation on the kinetics of activation. Unlike the type I cGKs (cGKIalpha and Ibeta), cGKII autophosphorylation did not alter the basal activity, nor the sensitivity of the enzyme to cyclic nucleotide activation. To determine residues responsible for autoinhibition of cGKII, Ala was substituted for basic residues (Lys(122), Arg(118), and Arg(119)) or a hydrophobic residue (Val(125)) within the putative pseudosubstrate domain of cGKII. The integrity of these residues was essential for full cGKII autoinhibition. Furthermore, a cGKII truncation mutant containing this autoinhibitory region demonstrated a nanomolar IC(50) toward a constitutively active form of cGKII. Finally, we present evidence that the dominant negative properties of this truncation mutant are specific to cGKII when compared with cAMP-dependent protein kinase Calpha and cGKIbeta. These findings extend the known differences in the activation mechanisms among cGK isoforms and allow the design of an isoform-specific cGKII inhibitor.     

Conserved alpha-helix acts as autoinhibitory sequence in AMP-activated protein kinase alpha subunits

AMP-activated protein kinase (AMPK) acts as an energy sensor, being activated by metabolic stresses and regulating cellular metabolism. AMPK is a heterotrimer consisting of a catalytic alpha subunit and two regulatory subunits, beta and gamma. It had been reported that the mammalian AMPK alpha subunit contained an autoinhibitory domain (alpha1: residues 313-392) and had little kinase activity. We have found that a conserved short segment of the alpha subunit (alpha1-(313-335)), which includes a predicted alpha-helix, is responsible for alpha subunit autoinhibition. The role of the residues in this segment for autoinhibition was further investigated by systematic site-directed mutation. Several hydrophobic and charged residues, in particular Leu-328, were found to be critical for alpha1 autoinhibition. An autoinhibitory structural model of human AMPK alpha1-(1-335) was constructed and revealed that Val-298 interacts with Leu-328 through hydrophobic bonding at a distance of about 4 A and may stabilize the autoinhibitory conformation. Further mutation analysis showed that V298G mutation significantly activated the kinase activity. Moreover, the phosphorylation level of acetyl-CoA carboxylase, the AMPK downstream substrate, was significantly increased in COS7 cells overexpressing AMPK alpha1-(1-394) with deletion of residues 313-335 (Deltaalpha394) and a V298G or L328Q mutation, and the glucose uptake was also significantly enhanced in HepG2 cells transiently transfected with Deltaalpha394, V298G, or L328Q mutants, which indicated that these AMPK alpha1 mutants are constitutively active in mammalian cells and that interaction between Leu-328 and Val-298 plays an important role in AMPK alpha autoinhibitory function.     

A conserved serine juxtaposed to the pseudosubstrate site of type I cGMP-dependent protein kinase contributes strongly to autoinhibition and lower cGMP affinity

Serines 64 and 79 are homologous residues that are juxtaposed to the autoinhibitory pseudosubstrate site in cGMP-dependent protein kinase type Ialpha and type Ibeta (PKG-Ialpha and PKG-Ibeta), respectively. Autophosphorylation of this residue is associated with activation of type I PKGs. To determine the role of this conserved serine, point mutations have been made in PKG-Ialpha (S64A, S64T, S64D, and S64N) and PKG-Ibeta (S79A). In wild-type PKG-Ialpha, basal kinase activity ratio (-cGMP/+cGMP) is 0.11, autophosphorylation increases this ratio 3-fold, and the K(a) and K(D) values for cGMP are 127 and 36 nm, respectively. S64A PKG-Ialpha basal kinase activity ratio increases 2-fold, cGMP binding affinity increases approximately 10-fold in both K(a) and K(D), and activation by autophosphorylation is slight. S64D and S64N mutants are nearly constitutively active in the absence of cGMP, cGMP binding affinity in each increases 18-fold, and autophosphorylation does not affect the kinase activity of these mutants. Mutation of the homologous site in PKG-Ibeta (S79A) increases the basal kinase activity ratio 2-fold and cGMP binding affinity 5-fold over that of wild-type PKG-Ibeta. The combined results demonstrate that a conserved serine juxtaposed to the pseudosubstrate site in type I PKGs contributes importantly to enzyme function by increasing autoinhibition and decreasing cGMP binding affinity.     

Identification of a conserved residue responsible for the autoinhibition of cGMP-dependent protein kinase Ialpha and beta

We isolated a constitutively active form of cGMP-dependent protein kinase Ialpha (cGK Ialpha) by PCR-driven random mutagenesis. The replacement of Ile-63 by Thr in the autoinhibitory domain results in the enhancement of autophosphorylation and the basal kinase activity in the absence of cGMP. The hydrophobicity at position 63 is essential for the inactive state of cGK Ialpha, and Ile-78 of cGK Ibeta is also required for the autoinhibitory property. Furthermore, cGK Ialpha (Ile-63-Thr) is constitutively active in vivo. These findings suggest that a conserved residue in the autoinhibitory domain was involved in the autoinhibition of both cGK Is.     

Regulatory mechanism of Dictyostelium myosin light chain kinase A

In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin-dependent protein kinase kinase as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr166 by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an approximately 140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by activated MLCK-A. Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr289 and that, subsequent to Thr166 phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Ser residues. Using site-directed mutagenesis, we show that autophosphorylation at Thr289 is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283-288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr166 phosphorylation. Simple removal of this region resulted in a significant increase in the kcat of MLCK-A; however, it did not generate maximum enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr166 phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme.     

Basic residues are important for Ca2+/calmodulin binding and activation but not autoinhibition of rabbit skeletal muscle myosin light chain kinase

Several allosterically modulated protein kinases have been shown to be regulated by an autoinhibitory domain located within the kinase molecules. The inhibitory domain has been proposed to act as a "pseudosubstrate" inhibitor binding to the substrate binding site of the kinase, thereby blocking the binding of the enzyme's true substrate. In this report, site-directed mutagenesis has been used to further investigate the mechanism of activation of the inhibitory domain of rabbit skeletal muscle myosin light chain kinase. Basic residues within the pseudosubstrate domain (572-573, 577-579, 580-581), which are analogous to the important substrate determinants of the myosin light chain, were found not to be required in order to maintain the kinase in an inhibited state. Two groups of these residues (577-579 and 581-582) were, however, found to be important for high affinity calmodulin binding to the kinase. These data suggest that the autoinhibitory domain of myosin light chain kinase may not function by directly mimicking the light chain substrate.     

Potent peptide inhibitors of smooth muscle myosin light chain kinase: mapping of the pseudosubstrate and calmodulin binding domains

Smooth muscle myosin light chain kinase (MLCK) is activated by calcium-calmodulin and, in turn, phosphorylates and activates the smooth muscle actomyosin ATPase, resulting in muscle contraction. The amino acid sequence of the regulatory domain of MLCK is known, and it contains a region that binds calmodulin and also bears a strong homology to the phosphorylation site in the substrate. Thus, it has been called the "pseudosubstrate". It has been proposed that calmodulin activates MLCK by binding to and reversing the autoinhibitory function of the pseudosubstrate. Synthetic peptides based on this sequence inhibit MLCK both by binding to calmodulin and by competing with the substrate at the active site. In the work reported here, we have synthesized a large number of peptides from the regulatory region of MLCK (MLCK 480-516). The region was systematically analyzed by dividing it into fragments of two to six amino acids, each containing one or more basic residues, in order to map in detail the calmodulin binding site and the autoinhibitory region. It was observed that both calmodulin binding and autoinhibition are mediated by several different fragments of the regulatory sequence. Two nonoverlapping peptides, MLCK 480-493 and MLCK 494-504, are similar in potency in inhibiting the enzyme (IC50's of 2 and 6 microM, respectively). Larger fragments, combining multiple inhibitory regions, are more potent inhibitors. For example, MLCK 480-504 is extremely potent, with an IC50 of 13 nM. The calmodulin binding site and active site directed inhibitory regions overlap, but are not identical. Residues 505-512 are important only for calmodulin binding.     

A recombinant form of the catalytic subunit of phosphorylase kinase that is soluble, monomeric, and includes key C-terminal residues.

Residues 302-326 of the catalytic (gamma) subunit of phosphorylase kinase (PhK) may comprise an autoinhibitory, pseudosubstrate domain that binds calmodulin. To study this, the cDNA corresponding to rabbit muscle PhKgamma was expressed using Escherichia coli. This yielded two stable, high-activity PhKgamma forms (35 and 42 kDa by SDS-PAGE) that were smaller than an authentic sample of rabbit muscle PhKgamma (45 kDa by SDS-PAGE). Each recombinant form was purified to homogeneity. The N-terminal sequence of the larger, 42-kDa form (pk42) matched that of the rabbit muscle enzyme. This suggested that pk42 consisted of PhKgamma residues 1-362, including the putative calmodulin-binding, autoinhibitory domain. Kinetic parameters obtained for pk42 were like those previously reported for the intact gamma subunit. This implied that the lack of 25 PhKgamma C-terminal residues did not affect phosphorylase kinase activity, but greatly improved enzyme stability. An additional 60 residues were removed from the C-terminus of pk42 using the protease m-calpain. This increased the kinase activity 1.5-fold. Consistent with this, the activity of a mutant PhKgamma that consisted of residues 1-300, denoted gamma1-300, was like that of the m-calpain-treated enzyme. Therefore, although the effect was small, some influence by the C-terminus of pk42 was noted. Moreover, when pk42 was incubated with ATP alone, a C-terminal threonine residue became phosphorylated. Although the influence of this autophosphorylation cannot be inferred from this data, it was evidence that the C-terminus accessed the enzyme's active site. Taken together, these data imply that pk42 will be useful to study phosphorylase kinase structure/activity relationships.     

Functional assembly of fragments from bisected smooth muscle myosin light chain kinase

The C-terminal regulatory segment of smooth muscle myosin light chain kinase folds back on its catalytic core to inhibit kinase activity. This regulatory segment consists of autoinhibitory residues linking the catalytic core to the calmodulin-binding sequence and perhaps additional C-terminal residues including an immunoglobulin-like motif. However, mutational and biochemical analyses showed no specific involvement of residues C-terminal to the calmodulin-binding sequence. To obtain additional insights on the proposed mechanisms for autoinhibition and Ca(2+)/calmodulin activation of the kinase, the polypeptide backbone chain of myosin light chain kinase was cleaved by genetic means to produce N- and C-terminal protein fragments. The N-terminal fragment containing the catalytic core was catalytically inactive when expressed alone. Co-expression of the N-terminal fragment with the C-terminal fragment containing the regulatory segment restored kinase activity. Deletion of the autoinhibitory linker residues without or with the calmodulin-binding sequence prevented restoration of kinase activity. In the presence or absence of Ca(2+)/calmodulin, regulatory segment binding occurred through the linker region connecting the catalytic core to the calmodulin-binding sequence. Collectively, these results indicate that residues C-terminal to the calmodulin-binding sequence (including the immunoglobulin-like motif) are not functional components of the regulatory segment. Furthermore, the principal autoinhibitory motif is contained in the sequence linking the catalytic core of myosin light chain kinase to the calmodulin-binding sequence.     

New insights into the autoinhibition mechanism of glycogen synthase kinase-3beta

It has been suggested that phosphorylation at serine 9 near the N-terminus of glycogen synthase kinase-3β (GSK-3β) mimics the prephosphorylation of its substrate and, therefore, the N-terminus functions as a pseudosubstrate. The molecular basis for the pseudosubstrate's binding to the catalytic core and autoinhibition has not been fully defined. Here, we combined biochemical and computational analyses to identify the potential residues within the N-terminus and the catalytic core engaged in autoinhibition of GSK-3β. Bioinformatic analysis found Arg4, Arg6, and Ser9 in the pseudosubstrate sequence to be extremely conserved through evolution. Mutations at Arg4 and Arg6 to alanine enhanced GSK-3β kinase activity and impaired its ability to autophosphorylate at Ser9. In addition, and unlike wild-type GSK-3β, these mutants were unable to undergo autoinhibition by phosphorylated Ser9. We further show that Gln89 and Asn95, located within the catalytic core, interact with the pseudosubstrate. Mutation at these sites prevented inhibition by phosphorylated Ser9. Furthermore, the respective mutants were not inhibited by a phosphorylated pseudosubstrate peptide inhibitor. Finally, computational docking of the pseudosubstrate into the catalytic active site of the kinase suggested specific interactions between Arg6 and Asn95 and of Arg4 to Asp181 (apart from the interaction of phosphorylated serine 9 with the “phosphate binding pocket”). Altogether, our study supports a model of GSK-3-pseudosubstrate autoregulation that involves phosphorylated Ser9, Arg4, and Arg6 within the N-terminus and identified the specific contact sites within the catalytic core.     

Small molecule antagonizes autoinhibition and activates AMP-activated protein kinase in cells

AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK alpha1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in alpha subunits, we screened a chemical library with inactive human alpha1(394) (alpha1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK alpha1(394), alpha1(335), alpha2(398), and even heterotrimer alpha1beta1gamma1. Based on PT1-docked AMPK alpha1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 with-out an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK.     

Structure of the complex of calmodulin with the target sequence of calmodulin-dependent protein kinase I: studies of the kinase activation mechanism

Calcium-saturated calmodulin (CaM) directly activates CaM-dependent protein kinase I (CaMKI) by binding to a region in the C-terminal regulatory sequence of the enzyme to relieve autoinhibition. The structure of CaM in a high-affinity complex with a 25-residue peptide of CaMKI (residues 294-318) has been determined by X-ray crystallography at 1.7 A resolution. Upon complex formation, the CaMKI peptide adopts an alpha-helical conformation, while changes in the CaM domain linker enable both its N- and C-domains to wrap around the peptide helix. Target peptide residues Trp-303 (interacting with the CaM C-domain) and Met-316 (with the CaM N-domain) define the mode of binding as 1-14. In addition, two basic patches on the peptide form complementary charge interactions with CaM. The CaM-peptide affinity is approximately 1 pM, compared with 30 nM for the CaM-kinase complex, indicating that activation of autoinhibited CaMKI by CaM requires a costly energetic disruption of the interactions between the CaM-binding sequence and the rest of the enzyme. We present biochemical and structural evidence indicating the involvement of both CaM domains in the activation process: while the C-domain exhibits tight binding toward the regulatory sequence, the N-domain is necessary for activation. Our crystal structure also enables us to identify the full CaM-binding sequence. Residues Lys-296 and Phe-298 from the target peptide interact directly with CaM, demonstrating overlap between the autoinhibitory and CaM-binding sequences. Thus, the kinase activation mechanism involves the binding of CaM to residues associated with the inhibitory pseudosubstrate sequence.     

Lack of constitutive activity of the free kinase domain of protein kinase C zeta. Dependence on transphosphorylation of the activation loop

Following the induction of apoptosis in mammalian cells, protein kinase C zeta (PKC zeta) is processed between the regulatory and catalytic domains by caspases, which increases its kinase activity. The catalytic domain fragments of PKC isoforms are considered to be constitutively active, because they lack the autoinhibitory amino-terminal regulatory domain, which includes a pseudosubstrate segment that plugs the active site. Phosphorylation of the activation loop at Thr(410) is known to be sufficient to activate the kinase function of full-length PKC zeta, apparently by inducing a conformational change, which displaces the amino-terminal pseudosubstrate segment from the active site. Amino acid substitutions for Thr(410) of the catalytic domain of PKC zeta (CAT zeta) essentially abolished the kinase function of ectopically expressed CAT zeta in mammalian cells. Similarly, substitution of Ala for a Phe of the docking motif for phosphoinositide-dependent kinase-1 prevented activation loop phosphorylation and abolished the kinase activity of CAT zeta. Treatment of purified CAT zeta with the catalytic subunit of protein phosphatase 1 decreased activation loop phosphorylation and kinase activity. Recombinant CAT zeta from bacteria lacked detectable kinase activity. Phosphoinositide-dependent kinase-1 phosphorylated the activation loop and activated recombinant CAT zeta from bacteria. Treatment of HeLa cells with fetal bovine serum markedly increased the phosphothreonine 410 content of CAT zeta and stimulated its kinase activity. These findings indicate that the catalytic domain of PKC zeta is intrinsically inactive and dependent on the transphosphorylation of the activation loop.     

Phosphorylation of Raf-1 by p21-activated kinase 1 and Src regulates Raf-1 autoinhibition

Exposure of cells to mitogens or growth factors stimulates Raf-1 activity through a complex mechanism that involves binding to active Ras, phosphorylation on multiple residues, and protein-protein interactions. Recently it was shown that the amino terminus of Raf-1 contains an autoregulatory domain that can inhibit its activity in Xenopus oocytes. In the present work we show that expression of the Raf-1 autoinhibitory domain blocks extracellular signal-regulated kinase 2 activation by the Raf-1 catalytic domain in mammalian cells. We also show that phosphorylation of Raf-1 on serine 338 by PAK1 and tyrosines 340 and 341 by Src relieves autoinhibition and that this occurs through a specific decrease in the binding of the Raf-1 regulatory domain to its catalytic domain. In addition, we demonstrate that phosphorylation of threonine 491 and serine 494, two phosphorylation sites in the catalytic domain that are required for Raf-1 activation, is unlikely to regulate autoinhibition. These results demonstrate that the autoinhibitory domain of Raf-1 is functional in mammalian cells and that its interaction with the Raf-1 catalytic domain is regulated by phosphorylation of serine 338 and tyrosines 340 and 341.     

B-Raf and Raf-1 are regulated by distinct autoregulatory mechanisms

B-Raf is a key regulator of the ERK pathway and is mutationally activated in two-thirds of human melanomas. In this work, we have investigated the activation mechanism of B-Raf and characterized the roles of Ras and of B-Raf phosphorylation in this regulation. Raf-1 is regulated by an N-terminal autoinhibitory domain whose actions are blocked by interaction with Ras and subsequent phosphorylation of Ser(338). We observed that B-Raf also contains an N-terminal autoinhibitory domain and that the interaction of this domain with the catalytic domain was inhibited by binding to active H-Ras. However, unlike Raf-1, the phosphorylation of B-Raf at Ser(445) was constitutive and was only moderately increased by expression of constitutively active H-Ras or constitutively active PAK1. Ser(445) phosphorylation is important to the B-Raf activation mechanism, however, because mutation of this site to alanine increased the affinity of the regulatory domain for the catalytic domain and increased autoinhibition. Similarly, expression of constitutively active PAK1 also decreased auto-inhibition. B-Raf autoinhibition was negatively regulated by acidic substitutions at phosphorylation sites within the activation loop of B-Raf and by the oncogenic substitution V599E. However, these substitutions did not affect the ability of the regulatory domain to co-immunoprecipitate with the catalytic domain. These data demonstrate that B-Raf activity is autoregulated, that constitutive phosphorylation of Ser(445) primes B-Raf for activation, and that a key feature of phosphorylation within the activation loop or of oncogenic mutations within this region is to block autoinhibition.     

An inhibitory segment of the catalytic subunit of phosphorylase kinase does not act as a pseudosubstrate.

The C terminus of the catalytic gamma subunit of phosphorylase kinase contains two autoinhibitory calmodulin binding domains designated PhK13 and PhK5. These peptides inhibit truncated gamma(1-300). Previous data show that PhK13 (residues 302-326) is a competitive inhibitor with respect to phosphorylase b, with a K(i) of 1.8 microm. This result suggests that PhK13 may bind to the active site of truncated gamma(1-300). Variants of PhK13 were prepared to localize the determinants for interaction with the catalytic fragment gamma(1-300). PhK13-1, containing residues 302-312, was found to be a competitive inhibitor with respect to phosphorylase b with a K(i) of 6.0 microm. PhK13 has been proposed to function as a pseudosubstrate inhibitor with Cys-308 occupying the site that normally accommodates the phosphorylatable serine in phosphorylase b. A PhK13-1 variant, C308S, was synthesized. Kinetic characterization of this peptide reveals that it does not serve as a substrate but is a competitive inhibitor. Additional variants were designed based on previous knowledge of phosphorylase kinase substrate determinants. Variants were analyzed as substrates and as inhibitors for truncated gamma(1-300). Although PhK13-1 does not appear to function as a pseudosubstrate, several specificity determinants employed in the recognition of phosphorylase b as substrate are utilized in the recognition of PhK13-1 as an inhibitor.