Regulation of calcineurin phosphatase activity by its autoinhibitory domain

The Ca(2+)-dependent activation of calcineurin phosphatase activity is regulated by an autoinhibitory element (residues 457-482) located 43 residues COOH-terminal of the calmodulin-binding domain (residues 390-414). Removal of residues 457-482 does not result in full Ca(2+)/calmodulin-independent activity. Full activity in the absence of Ca(2+) requires the removal of residues 420-457. In the present study the presence of additional autoinhibitory elements within residues 420-457 was tested using two calcineurin A subunit COOH-terminal region constructs containing residues 420-511 (AI(420-511)) or 328-511 (AI(328-511)). Using recombinant, Ca(2+)/calmodulin-independent calcineurin, AI(420-511) and AI(328-511) were three- to fourfold more potent inhibitors of calcineurin phosphatase activity than the synthetic calcineurin autoinhibitory peptide(457-482). Calmodulin reversed the inhibition of calcineurin phosphatase activity by AI(328-511) but not AI(420-511). Kinetic studies indicated that AI(420-511) exhibited mixed-type inhibition and that the enzyme/substrate/inhibitor complex is partially active. These results indicate that (i) additional autoinhibitory elements are present within residues 420-457, (ii) calmodulin-binding to the autoinhibitory domain neutralizes the inhibitory function of the 420-457 autoinhibitory segment, (iii) the full-length autoinhibitory domain is a mixed-type inhibitor of calcineurin phosphatase activity, and (iv) the enzyme/substrate/inhibitor complex is partially catalytically active.     

Regulation of PTEN activity by its carboxyl-terminal autoinhibitory domain

The regulation of PTEN intrinsic biochemical properties has not been fully elucidated. In this report, we investigated the role of the PTEN carboxyl-terminal tail domain in regulating its membrane targeting and catalytic functions. Characterization of a panel of PTEN phosphorylation site mutants revealed that mutating Ser-385 to alanine (S385A) promoted membrane localization in vivo and phosphatase activity in vitro. Furthermore, S385A mutation was associated with a substantial reduction in the phosphorylation of the Ser-380/Thr-382/Thr-383 cluster. Therefore, Ser-385 could prime additional dephosphorylation events to regulate PTEN catalytic activity. Moreover, substituting Ser-380/Thr-382/Thr-383 to phosphomimic residues reversed the phosphatase activity of the S385A mutation. Next, we further defined the underlying mechanisms responsible for the COOH-terminal tail region in modulating PTEN biological activity. We have identified an interaction between the 71-amino acid carboxyl-terminal tail region and the CBRIII motif of the C2 domain, which has been implicated in membrane binding. In addition, a synthetic phosphomimic peptide encompassing the phosphorylation site cluster between amino acids 368 and 390 within the tail region mediated the suppression of PTEN catalytic activity in vitro. This same peptide when expressed in cultured cells also impeded PTEN membrane localization and enhanced phospho-Akt levels. Thus, our data suggest that the COOH-terminal tail can act as an autoinhibitory domain to control both PTEN membrane recruitment and phosphatase activity.     

Regulation of focal adhesion kinase by its amino-terminal domain through an autoinhibitory interaction

We have investigated a role for the amino-terminal FERM-like domain of the focal adhesion kinase (FAK) as a negative regulator of its own activity and phosphorylation state. Deletion of the first 375 amino acids from the amino terminus of FAK increases its catalytic activity in vitro, its phosphorylation when expressed in mammalian cells, and the phosphorylation of a FAK substrate, paxillin. Deletion mutants are phosphorylated in suspension, suggesting that they are no longer regulated by adhesion. The amino terminus of FAK can interact with the kinase domain of FAK in vitro and in vivo, suggesting that it might act as an autoinhibitor of FAK activity. The amino terminus of FAK can act in trans to inhibit FAK phosphorylation when expressed in mammalian cells or to directly inhibit FAK activity in vitro. Expression of the amino terminus of FAK inhibits cell cycle progression in CHO cells, consistent with its inhibition of FAK phosphorylation and function in trans. A glutathione S-transferase fusion protein containing the cytoplasmic tail of the beta1 integrin stimulates FAK activity in vitro, suggesting that FAK could be regulated by molecular interactions with the amino terminus. Based on these and previous data, we propose a working model for activation of FAK in cell adhesion.     

Identification of an autoinhibitory domain in calcineurin

The hypothesis that calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, contains an autoinhibitory domain was tested using synthetic peptides corresponding to regions of the carboxyl-terminus of calcineurin. Of the several peptides analyzed, one, containing residues I-T-S-F-E-E-A-K-G-L-D-R-I-N-E-R-M-P-P-R-R-D-A-M-P, gave complete inhibition of its protein phosphatase activity. Using [32P]myosin light chain as substrate an IC50 of about 10 microM was obtained with either native calcineurin, assayed in the presence of Ca2+/calmodulin, or with calcineurin subjected to partial proteolysis which converts it to a fully active phosphatase when assayed in the presence of [ethylenebis (oxyethylenenitrilo)]tetraacetic acid. With 50 mM p-nitrophenylphosphate as substrate an IC50 of about 40 microM was observed. Studies with overlapping peptides suggested that the sequence P-P-R-R-D-A-M-P was essential but not sufficient for the observed inhibition. Kinetic analysis indicated that the inhibition of phosphatase activity was not competitive with respect to [32P]myosin light chain. This peptide did not show significant inhibition of the catalytic subunits of protein phosphatases type I or type IIA or of Ca2+/calmodulin-dependent protein kinase II. These results indicate that amino acids within this sequence of calcineurin constitute a unique autoinhibitory domain which interacts with the active site and is responsible for the low basal phosphatase activity in the absence of Ca2+/calmodulin.     

Regulation of MTK1/MEKK4 kinase activity by its N-terminal autoinhibitory domain and GADD45 binding

A variety of cellular stresses activate the stress-responsive mitogen-activated protein (MAP) kinases p38 and JNK. In this study, we studied the activation mechanism of a human MAP kinase kinase kinase, MTK1 (also known as MEKK4), which mediates activation of both p38 and JNK. MTK1 has an extensive N-terminal noncatalytic domain composed of approximately 1,300 amino acids. Full-length or near full-length MTK1 is catalytically inactive when expressed in Saccharomyces cerevisiae cells, as it is in mammalian cells. Deletion of a segment including positions 253 to 553 activates kinase, indicating that this segment contains the autoinhibitory domain. In the autoinhibited conformation, the MTK1 kinase domain cannot interact with its substrate, MKK6. By a functional complementation screening with yeast cells, GADD45 proteins (GADD45alpha, beta, and gamma) were identified as MTK1 activators. GADD45 proteins bind a site in MTK1 near the inhibitory domain and relieve autoinhibition. Mutants of full-length MTK1 were isolated that can interact with MKK6 in the absence of the activator GADD45 proteins. These MTK1 mutants are constitutively active, in both yeast and mammalian cells. A model of MTK1 autoinhibition by the N-terminal inhibitory domain and activation by GADD45 binding is presented.     

Regulation of GTPase function by autophosphorylation

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Regulation of human cystathionine beta-synthase by S-adenosyl-L-methionine: evidence for two catalytically active conformations involving an autoinhibitory domain in the C-terminal region

Cystathionine beta-synthase (CBS), condensing homocysteine and serine, represents a key regulatory point in the biosynthesis of cysteine via the transsulfuration pathway. Inherited deficiency of CBS causes homocystinuria. CBS is activated by S-adenosyl-L-methionine (AdoMet) by inducing a conformational change involving a noncatalytic C-terminal region spanning residues 414-551. We report the purification of two patient-derived C-terminal mutant forms of CBS, S466L and I435T, that provide new insight into the mechanism of CBS regulation and indicate a regulatory function for the "CBS domain". Both of these point mutations confer catalytically active proteins. The I435T protein is AdoMet inducible but is 10-fold less responsive than wild-type (WT) CBS to physiologically relevant concentrations of this compound. The S466L form does not respond to AdoMet but is constitutively activated to a level intermediate between those of WT CBS in the presence and absence of AdoMet. Both mutant proteins are able to bind AdoMet, indicating that their impairment is related to their ability to assume the fully activated conformation that AdoMet induces in WT CBS. We found that I435T and WT CBS can be activated by partial thermal denaturation but that the AdoMet-stimulated WT, S466L, and a truncated form of CBS lacking the C-terminal region cannot be further activated by this treatment. Tryptophan and PLP fluorescence data for these different forms of CBS indicate that activation by AdoMet, limited proteolysis, and thermal denaturation share a common mechanism involving the displacement of an autoinhibitory domain located in the C-terminal region of the protein.     

Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress

Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane-coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress.     

Identification of an autoinhibitory domain in the insulin receptor tyrosine kinase

We have tested the hypothesis that activation of the insulin receptor tyrosine kinase is due to autophosphorylation of tyrosines 1146, 1150 and 1151 within a putative autoinhibitory domain. A synthetic peptide corresponding to residues 1134–1162, with tyrosines substituted by alanine or phenylalanine, of the insulin receptor subunit was tested for its inhibitory potency and specificity towards the tyrosine kinase activity. This synthetic peptide gave inhibition of the insulin receptor tyrosine kinase autophosphorylation and phosphorylation of the exogenous substrate poly(Glu, Tyr) with an approximate IC₅₀ of 100 M. Inhibition appeared to be independent of the concentrations of insulin or the substrate poly(Glu, Tyr) but was decreased by increasing concentrations of ATP. This same peptide also inhibited the EGF receptor tyrosine kinase but not a serine/threonine protein kinase. These results are consistent with the hypothesis that this autophosphorylation domain contains an autoinhibitory sequence. (Mol Cell Biochem120: 103–110, 1993)Abbreviations IR Insulin Receptor - SDS/PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis - CaM Calmodulin - HEPES 4-(2-Hydroxyethyl)-Piperazineethane-Sulfonic Acid - DMEM Dulbecco's Modified Eagle' Medium - PMSF Phenylmethyl-Sulfonyl Fluoride - HPLC High Performance Liquid Chromatography - PKC Protein Kinase C - PKI Inhibitory Peptide for cAMP-Kinase - CaMK II Ca²⁺/Calmodulin-Dependent Protein Kinase II - CaN A A Subunit of Calcineurin     

The C-terminal domain of 4-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii is an autoinhibitory domain

p-Hydroxyphenylacetate (HPA) 3-hydroxylase from Acinetobacter baumannii consists of a reductase component (C(1)) and an oxygenase component (C(2)). C(1) catalyzes the reduction of FMN by NADH to provide FMNH(-) as a substrate for C(2). The rate of reduction of flavin is enhanced ～20-fold by binding HPA. The N-terminal domain of C(1) is homologous to other flavin reductases, whereas the C-terminal domain (residues 192-315) is similar to MarR, a repressor protein involved in bacterial antibiotic resistance. In this study, three forms of truncated C(1) variants and single site mutation variants of residues Arg-21, Phe-216, Arg-217, Ile-246, and Arg-247 were constructed to investigate the role of the C-terminal domain in regulating C(1). In the absence of HPA, the C(1) variant in which residues 179-315 were removed (t178C(1)) was reduced by NADH and released FMNH(-) at the same rates as wild-type enzyme carries out these functions in the presence of HPA. In contrast, variants with residues 231-315 removed behaved similarly to the wild-type enzyme. Thus, residues 179-230 are involved in repressing the production of FMNH(-) in the absence of HPA. These results are consistent with the C-terminal domain in the wild-type enzyme being an autoinhibitory domain that upon binding the effector HPA undergoes conformational changes to allow faster flavin reduction and release. Most of the single site variants investigated had catalytic properties similar to those of the wild-type enzyme except for the F216A variant, which had a rate of reduction that was not stimulated by HPA. F216A could be involved with HPA binding or in the required conformational change for stimulation of flavin reduction by HPA.     

WNK1 and WNK4 modulate CFTR activity

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated chloride channel. WNK kinases are widely expressed modulators of ion transport. WNK1 and WNK4, two WNK kinases that are mutated in familial hyperkalemic hypertension (FHHt), are co-expressed with CFTR in several organs, raising the possibility that WNK kinases might alter CFTR activity in vivo or that CFTR could be involved in the pathogenesis of FHHt. Here, we report that WNK1 co-localizes with CFTR protein in pulmonary epithelial cells. Co-expression of WNK1 or WNK4 with CFTR in Xenopus laevis oocytes suppresses chloride channel activity. The effect of WNK4 is dose dependent and occurs, at least in part, by reducing CFTR protein abundance at the plasma membrane. This effect is independent of WNK4 kinase activity. In contrast, the effect of WNK1 on CFTR activity requires intact WNK1 kinase activity. Moreover WNK1 and WNK4 exhibit additive CFTR inhibition. Previous reports suggest that patients with FHHt exhibit mild changes in nasal potential difference that resemble the more severe changes that occur in cystic fibrosis. We report that the FHHt-causing mutant WNK4 Q562E is a more potent inhibitor of CFTR activity than is the wild-type WNK4. Taken together, these results suggest that WNK1 and WNK4 may modulate CFTR activity; they further suggest that WNK kinases may be potential therapeutic targets for cystic fibrosis.     

Identification of an autoinhibitory domain of p21-activated protein kinase 5

The p21-activated protein kinases (Paks) are serine/threonine protein kinases activated by binding to Rho family small GTPases, Rac and Cdc42. Recently, Pak family members have been subdivided into two groups, I and II. Group II Paks, including Pak4, Pak5, and Pak6, does not contain the highly conserved autoinhibitory domain that is found in the group I Paks members, i.e. Pak1, Pak2, and Pak3. In the present study, we have purified the glutathione S-transferase fusion form of Pak5 and shown for the first time that Pak5 autophosphorylation can be activated by GTP bound form of Cdc42. Mutation of histidine residues 19 and 22 to leucine on the p21-binding domain of Pak5 completely abolished the binding of Cdc42 and the Cdc42-mediated autophosphorylation. On the other hand, mutation of tyrosine 40 to cysteine of Cdc42 did not knockout the binding of Pak5. Analysis of C-terminal deletion mutants has identified an autoinhibitory fragment of Pak5 that is absent from other group II Pak family members. Taken together, these results suggest that Pak5, like Pak1, contains an autoinhibitory domain and its activity is regulated by Cdc42.     

WNK1 is an unexpected autophagy inhibitor

Autophagy is a cellular degradation pathway that is essential to maintain cellular physiology, and deregulation of autophagy leads to multiple diseases in humans. In a recent study, we discovered that the protein kinase WNK1 (WNK lysine deficient protein kinase 1) is an inhibitor of autophagy. The loss of WNK1 increases both basal and starvation-induced autophagy. In addition, the depletion of WNK1 increases the activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex, which is required to induce autophagy. Moreover, the loss of WNK1 increases the expression of ULK1 (unc-51 like kinase 1), which is upstream of the PtdIns3K complex. It also increases the pro-autophagic phosphorylation of ULK1 at Ser555 and the activation of AMPK (AMP-activated protein kinase), which is responsible for that phosphorylation. The inhibition of AMPK by compound C decreases the magnitude of autophagy induction following WNK1 loss; however, it does not prevent autophagy induction. We found that the UVRAG (UV radiation resistance associated gene), which is a component of the PtdIns3K, binds to the N-terminal region of WNK1. Moreover, WNK1 partially colocalizes with UVRAG and this colocalization decreases when autophagy is stimulated in cells. The loss of WNK1 also alters the cellular distribution of UVRAG. The depletion of the downstream target of WNK1, OXSR1/OSR1 (oxidative-stress responsive 1) has no effect on autophagy, whereas the depletion of its relative STK39/SPAK (serine/threonine kinase 39) induces autophagy under nutrient-rich and starved conditions.     

Adenylyl cyclase Rv1264 from Mycobacterium tuberculosis has an autoinhibitory N-terminal domain

Mycobacterium tuberculosis contains 15 class III adenylyl cyclase genes. The gene Rv1264 is predicted to be composed of two distinct protein modules. The C terminus seems to code for a catalytic domain belonging to a subfamily of adenylyl cyclase isozymes mostly found in Gram-positive bacteria. The expressed protein was shown to function as a homodimeric adenylyl cyclase (1 micromol of cAMP x mg(-1) x min(-1)). In analogy to the structure of the mammalian adenylyl cyclase catalyst, six amino acids were targeted by point mutations and found to be essential for catalysis. The N-terminal region represents a novel protein domain, the occurrence of which is restricted to several adenylyl cyclases present in Gram-positive bacteria. The purified full-length enzyme was 300-fold less active than the catalytic domain alone. Thus, the N-terminal domain appeared to be autoinhibitory. The N-terminal domain contains three prominent polar amino acid residues (Asp(107), Arg(132), and Arg(191)) that are invariant in all seven sequences of this domain currently available. Mutation of Asp(107) to Ala relaxed the inhibition and resulted in a 6-fold increase in activity of the Rv1264 holoenzyme, thus supporting the role of this domain as a potential novel regulator of adenylyl cyclase activity.     

Regulation of Chk1 by its C-terminal domain

Chk1 is a protein kinase that is the effector molecule in the G2 DNA damage checkpoint. Chk1 homologues have an N-terminal kinase domain, and a C-terminal domain of ~200 amino acids that contains activating phosphorylation sites for the ATM/R kinases, though the mechanism of activation remains unknown. Structural studies of the human Chk1 kinase domain show an open conformation; the activity of the kinase domain alone is substantially higher in vitro than full-length Chk1, and coimmunoprecipitation studies suggest the C-terminal domain may contain an autoinhibitory activity. However, we show that truncation of the C-terminal domain inactivates Chk1 in vivo. We identify additional mutations within the C-terminal domain that activate ectopically expressed Chk1 without the need for activating phosphorylation. When expressed from the endogenous locus, activated alleles show a temperature-sensitive loss of function, suggesting these mutations confer a semiactive state to the protein. Intragenic suppressors of these activated alleles cluster to regions in the catalytic domain on the face of the protein that interacts with substrate, suggesting these are the regions that interact with the C-terminal domain. Thus, rather than being an autoinhibitory domain, the C-terminus of Chk1 also contains domains critical for adopting an active configuration.     

Comparison of WNK4 and WNK1 kinase and inhibiting activities

WNK kinases are novel serine/threonine protein kinases. Mutations in two members of the WNK family, WNK1 and WNK4, cause familial hyperkalemic hypertension. These kinases regulate ion transport across diverse epithelia; WNK4 reduces activity of the Na-Cl cotransporter activity and the potassium channel, ROMK, by reducing their appearance at the plasma membrane. We examined the kinase activity of WNK1 and WNK4 in vitro. A glutathione S-transferase (GST) fusion protein of the WNK1 kinse domain phosphorylated itself and a substrate protein, as reported previously. A longer construct, containing the autoinhibitory domain, did not. A GST WNK4 kinase domain construct demonstrated no kinase activity, in vitro or in HEK 293 cells. WNK4 constructs that included a region homologous to the autoinhibitory domain of WNK1 inhibited WNK1 kinase activity. Inhibition by a short WNK4 segment, WNK4 (444-518), was greater than inhibition by WNK4 (444-563). Together, these results suggest that WNK4 must be activated by currently unknown factors to exhibit kinase activity and that WNK4 contains an inhibitory domain that can inhibit the kinase activity of WNK1.     

UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBDelta4) leads to multiple changes in protein function. Protein dimerization decreases with an approximately 8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBDelta4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBDelta4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.     

Regulation of the nonreceptor tyrosine kinase Brk by autophosphorylation and by autoinhibition

Brk (breast tumor kinase) is a nonreceptor tyrosine kinase that is most closely related to the Frk family of kinases, and more distantly to Src family kinases. Brk was originally identified in a screen for tyrosine kinases that are overexpressed in human metastatic breast tumors. To shed light on the activity and regulation of Brk and related tyrosine kinases, we expressed and purified Brk using the Sf9/baculovirus system. We characterized the substrate specificity of Brk using synthetic peptides, and we show that the kinetic parameters K(m) and k(cat) both play a role in specificity. We carried out mass spectrometry experiments to show that Brk autophosphorylates within the predicted kinase activation loop and at additional sites in the N terminus. Autophosphorylation increases enzyme activity of wild-type Brk but not of a Y342A mutant form of Brk. We also carried out experiments to address the possible involvement of the Src homology (SH) 2 and SH3 domains of Brk in enzyme regulation. Mutation of a C-terminal tyrosine (Tyr-447) increases enzyme activity and SH2 domain accessibility, consistent with a role for this residue in autoinhibition. A proline-rich peptide activates Brk, suggesting that the SH3 domain is also involved in maintaining an inactive form of Brk. These biochemical results for Brk may aid in the understanding of other tyrosine kinases in the Frk family.     

Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes

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