Molecular mechanisms underlying KVS-1-MPS-1 complex assembly

Formation of heteromeric complexes between voltage-gated K(+) (Kv) channels and accessory (beta) subunits is a widespread means to generate heterogeneity of K(+) current in the nervous system. Here we investigate the principles that determine the interactions of Caenorhabditis elegans MPS-1, a bifunctional beta-subunit that possesses kinase activity, with Kv channels. MPS-1 belongs to the evolutionarily conserved family of KCNE beta-subunits that modulate the functional properties of a variety of Kv channels and that, when defective, can cause congenital and acquired disease in Homo sapiens. In Chinese hamster ovary cells, MPS-1 forms stable complexes with different alpha-subunits. The transmembrane domain of MPS-1 is necessary and sufficient for MPS-1 complex formation. The hydropathicity of the transmembrane domain is an important factor controlling MPS-1 assembly. A highly hydrophobic MPS-1 mutant fails to interact with its endogenous channel partners when transgenically expressed in living worms. The hydropathic mechanism does not require specific points of contact between interacting proteins. This may allow MPS-1 to assemble with various Kv channels, presumably modifying the electrical properties of each.     

Structure of a human A-type potassium channel interacting protein DPPX, a member of the dipeptidyl aminopeptidase family

It has recently been reported that dipeptidyl aminopeptidase X (DPPX) interacts with the voltage-gated potassium channel Kv4 and that co-expression of DPPX together with Kv4 pore forming alpha-subunits, and potassium channel interacting proteins (KChIPs), reconstitutes properties of native A-type potassium channels in vitro. Here we report the X-ray crystal structure of the extracellular domain of human DPPX determined at 3.0A resolution. This structure reveals the potential for a surface electrostatic change based on the protonation state of histidine. Subtle changes in extracellular pH might modulate the interaction of DPPX with Kv4.2 and possibly with other proteins. We propose models of DPPX interaction with the voltage-gated potassium channel complex. The dimeric structure of DPPX is highly homologous to the related protein DPP-IV. Comparison of the active sites of DPPX and DPP-IV reveals loss of the catalytic serine residue but the presence of an additional serine near the "active" site. However, the arrangement of residues is inconsistent with that of canonical serine proteases and DPPX is unlikely to function as a protease (dipeptidyl aminopeptidase).     

Structural insights into the autoactivation mechanism of p21-activated protein kinase

p21-activated kinases (PAKs) play an important role in diverse cellular processes. Full activation of PAKs requires autophosphorylation of a critical threonine/serine located in the activation loop of the kinase domain. Here we report crystal structures of the phosphorylated and unphosphorylated PAK1 kinase domain. The phosphorylated PAK1 kinase domain has a conformation typical of all active protein kinases. Interestingly, the structure of the unphosphorylated PAK1 kinase domain reveals an unusual dimeric arrangement expected in an authentic enzyme-substrate complex, in which the activation loop of the putative "substrate" is projected into the active site of the "enzyme." The enzyme is bound to AMP-PNP and has an active conformation, whereas the substrate is empty and adopts an inactive conformation. Thus, the structure of the asymmetric homodimer mimics a trans-autophosphorylation complex, and suggests that unphosphorylated PAK1 could dynamically adopt both the active and inactive conformations in solution.     

Structure of dystrophia myotonica protein kinase

Dystrophia myotonica protein kinase (DMPK) is a serine/threonine kinase composed of a kinase domain and a coiled‐coil domain involved in the multimerization. The crystal structure of the kinase domain of DMPK bound to the inhibitor bisindolylmaleimide VIII (BIM‐8) revealed a dimeric enzyme associated by a conserved dimerization domain. The affinity of dimerisation suggested that the kinase domain alone is insufficient for dimerisation in vivo and that the coiled‐coil domains are required for stable dimer formation. The kinase domain is in an active conformation, with a fully‐ordered and correctly positioned αC helix, and catalytic residues in a conformation competent for catalysis. The conserved hydrophobic motif at the C‐terminal extension of the kinase domain is bound to the N‐terminal lobe of the kinase domain, despite being unphosphorylated. Differences in the arrangement of the C‐terminal extension compared to the closely related Rho‐associated kinases include an altered PXXP motif, a different conformation and binding arrangement for the turn motif, and a different location for the conserved NFD motif. The BIM‐8 inhibitor occupies the ATP site and has similar binding mode as observed in PDK1.     

Biogenesis of the T1-S1 linker of voltage-gated K+ channels

In the model derived from the crystal structure of Kv1.2, a six-transmembrane voltage-gated potassium channel, the linker between a cytosolic tetramerization domain, T1, and the first transmembrane segment, S1, is projected radially outward from the channel's central axis. This T1-S1 linker was modeled as two polyglycine helices to accommodate the residues between T1 and S1 [Long et al. (2005) Science 309, 897-903]; however, the structure of this linker is not known. Here, we investigate whether a compact secondary structure of the T1-S1 linker exists at an early stage of Kv channel biogenesis. We have used a mass-tagging accessibility assay to report the biogenesis of secondary structure for three consecutive regions of Kv1.3, a highly homologous isoform of Kv1.2. The three regions include the T1-S1 linker and its two flanking regions, alpha5 of the T1 domain and S1. Both alpha5 and S1 manifest compact structures (helical) inside the ribosomal exit tunnel, whereas the T1-S1 linker does not. Moreover, the location of the peptide in the tunnel influences compaction.     

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.     

The Src homology 3 domain of the beta-subunit of voltage-gated calcium channels promotes endocytosis via dynamin interaction

High voltage-gated calcium channels enable calcium entry into cells in response to membrane depolarization. Association of the auxiliary beta-subunit to the alpha-interaction-domain in the pore-forming alpha1-subunit is required to form functional channels. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 and a guanylate kinase domain. Although the latter is responsible for the high affinity binding to the alpha-interaction domain, the functional significance of the Src homology 3 domain remains elusive. Here, we show that injection of isolated beta-subunit Src homology 3 domain into Xenopus laevis oocytes expressing the alpha1-subunit reduces the number of channels in the plasma membrane. This effect is reverted by coexpressing alpha1 with a dominant-negative mutant of dynamin, a GTPase involved in receptor-mediated endocytosis. Full-length beta-subunit also down-regulates voltage-gated calcium channels but only when lacking the alpha-interaction domain. Moreover, isolated Src homology 3 domain and the full-length beta-subunit were found to interact in vitro with dynamin and to internalize the distantly related Shaker potassium channel. These results demonstrate that the beta-subunit regulates the turnover of voltage-gated calcium channels and other proteins in the cell membrane. This effect is mediated by dynamin and depends on the association state of the beta-subunit to the alpha1-pore-forming subunit. Our findings define a novel function for the beta-subunit through its Src homology 3 domain and establish a link between voltage-gated calcium channel activity and the cell endocytic machinery.     

Calphostin-C stimulates epidermal growth factor receptor phosphorylation and internalization via light-dependent mechanism

Calphostin-C with perylenequinone structure is known to bind the regulatory domain of protein kinase C (PKC) and to inhibit kinase activity in vitro in a light-dependent fashion. We have found that calphostin-C induces substantial serine and threonine phosphorylation of the epidermal growth factor (EGF) receptor in a light-dependent fashion in the EGF receptor-hyperproducing squamous carcinoma cell line NA. Tryptic phospho-peptide mapping and phospho-amino acid analysis revealed that calphostin-C–-enhanced phosphorylation was on threonine 669, serine 671, serine 1046/1047, and serine 1166. However, caiphostin-C did not inhibit phosphorylation of the 80 K protein, a cytosolic major substrate of PKC (MARCKS). Staurosporine, a potent PKC inhibitor with affinity for the catalytic domain of PKC, inhibited phosphorylation of the 80 K protein and 12-O-tetradecanoyl-13-phorbol acetate induction of EGF receptor phosphorylation but did not inhibit the calphostin-C induction of the EGF receptor phosphorylation. These results suggest that the target of calphostin-C in vivo is different from that of staurosporine and thus calphostin-C in vivo does not inhibit PKC. Furthermore, calphostin-C enhanced the internalization of phosphorylated EGF receptor. Thus, calphostin-C apparently activates a novel signal transduction pathway which involves phosphorylation and internalization of the EGF receptor via light-dependent mechanism. © 1994 Wiley-Liss, Inc.     

WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II

We have cloned and characterized a novel mammalian serine/threonine protein kinase WNK1 (with no lysine (K)) from a rat brain cDNA library. WNK1 has 2126 amino acids and can be detected as a protein of approximately 230 kDa in various cell lines and rat tissues. WNK1 contains a small N-terminal domain followed by the kinase domain and a long C-terminal tail. The WNK1 kinase domain has the greatest similarity to the MEKK protein kinase family. However, overexpression of WNK1 in HEK293 cells exerts no detectable effect on the activity of known, co-transfected mitogen-activated protein kinases, suggesting that it belongs to a distinct pathway. WNK1 phosphorylates the exogenous substrate myelin basic protein as well as itself mostly on serine residues, confirming that it is a serine/threonine protein kinase. The demonstration of activity was striking because WNK1, and its homologs in other organisms lack the invariant catalytic lysine in subdomain II of protein kinases that is crucial for binding to ATP. A model of WNK1 using the structure of cAMP-dependent protein kinase suggests that lysine 233 in kinase subdomain I may provide this function. Mutation of this lysine residue to methionine eliminates WNK1 activity, consistent with the conclusion that it is required for catalysis. This distinct organization of catalytic residues indicates that WNK1 belongs to a novel family of serine/threonine protein kinases.     

The TRPM7 channel is inactivated by PIP(2) hydrolysis

TRPM7 (ChaK1, TRP-PLIK, LTRPC7) is a ubiquitous, calcium-permeant ion channel that is unique in being both an ion channel and a serine/threonine kinase. The kinase domain of TRPM7 directly associates with the C2 domain of phospholipase C (PLC). Here, we show that in native cardiac cells and heterologous expression systems, G alpha q-linked receptors or tyrosine kinase receptors that activate PLC potently inhibit channel activity. Numerous experimental approaches demonstrated that phosphatidylinositol 4,5-bisphosphate (PIP(2)), the substrate of PLC, is a key regulator of TRPM7. We conclude that receptor-mediated activation of PLC results in the hydrolysis of localized PIP(2), leading to inactivation of the TRPM7 channel.     

NMR structure of the "ball-and-chain" domain of KCNMB2, the beta 2-subunit of large conductance Ca2+- and voltage-activated potassium channels

The auxiliary beta-subunit KCNMB2 (beta(2)) endows the non-inactivating large conductance Ca(2+)- and voltage-dependent potassium (BK) channel with fast inactivation. This process is mediated by the N terminus of KCNMB2 and closely resembles the "ball-and-chain"-type inactivation observed in voltage-gated potassium channels. Here we investigated the solution structure and function of the KCNMB2 N terminus (amino acids 1-45, BKbeta(2)N) using NMR spectroscopy and patch clamp recordings. BKbeta(2)N completely inactivated BK channels when applied to the cytoplasmic side; its interaction with the BK alpha-subunit is characterized by a particularly slow dissociation rate and an affinity in the upper nanomolar range. The BKbeta(2)N structure comprises two domains connected by a flexible linker: the pore-blocking "ball domain" (formed by residues 1-17) and the "chain domain" (between residues 20-45) linking it to the membrane segment of KCNMB2. The ball domain is made up of a flexible N terminus anchored at a well ordered loop-helix motif. The chain domain consists of a 4-turn helix with an unfolded linker at its C terminus. These structural properties explain the functional characteristics of BKbeta(2)N-mediated inactivation.     

Dual specificity protein kinase activity of testis-specific protein kinase 1 and its regulation by autophosphorylation of serine-215 within the activation loop

TESK1 (testis-specific protein kinase 1) is a protein kinase with a structure composed of an N-terminal protein kinase domain and a C-terminal proline-rich domain. Whereas the 3.6-kilobase TESK1 mRNA is expressed predominantly in the testis, a faint 2.5-kilobase TESK1 mRNA is expressed ubiquitously. The kinase domain of TESK1 contains in the catalytic loop in subdomain VIB an unusual DLTSKN sequence, which is not related to the consensus sequence of either serine/threonine kinases or tyrosine kinases. In this study, we show that TESK1 has kinase activity with dual specificity on both serine/threonine and tyrosine residues. In an in vitro kinase reaction, the kinase domain of TESK1 underwent autophosphorylation on serine and tyrosine residues and catalyzed phosphorylation of histone H3 and myelin basic protein on serine, threonine, and tyrosine residues. Site-directed mutagenesis analyses revealed that Ser-215 within the "activation loop" of the kinase domain is the site of serine autophosphorylation of TESK1. Replacement of Ser-215 by alanine almost completely abolished serine autophosphorylation and histone H3 kinase activities. In contrast, replacement of Ser-215 by glutamic acid abolished serine autophosphorylation activity but retained histone H3 kinase activity. These results suggest that autophosphorylation of Ser-215 is an important step to positively regulate the kinase activity of TESK1.     

Activity and substrate specificity of the murine STK2 Serine/Threonine kinase that is structurally related to the mitotic regulator protein NIMA of Aspergillus nidulans.

We isolated a murine STK2 (mSTK2) cDNA that is homologous to murine Nek1 serine/threonine kinase, a family member related to the cell cycle regulator kinase NIMA of Aspergillus nidulans. Structural comparison demonstrated that the kinase domain of mSTK2 is highly similar to NIMA/Nek family but the C-terminal region is not similar to any proteins except for human STK2 (hSTK2). Similarly to Nek1, mSTK2 is expressed ubiquitously among various organs and is upregulated in the testis. The expression and localization of mSTK2 are not associated with the cell cycle progression of mitogen-activated lymphocyte and DNA-transfected fibroblast. The substrate specificity of mSTK2 is similar to NIMA, but the phosphorylation is observed exclusively upon threonine residues rather than serine. The mSTK2 is shown to be a new member of the NIMA/Nek family with similar substrate specificity, which might participate in a different role from NIMA kinase involved in the cell cycle regulation.     

Structural and functional studies of casein kinase I-like protein from rice

Casein kinase I (CKI) is a protein serine/threonine kinase that is highly conserved from plants to animals. It performs various functions in both the cytoplasm and nucleus, such as DNA repair, cell cycle, cytokinesis, vesicular trafficking, morphogenesis and circadian rhythm. CKI proteins contain a highly conserved kinase domain responsible for catalytic activity at the N-terminus and a highly diverse regulatory domain responsible for determining substrate specificity at the C-terminus. CKI-like protein has been identified in plants, including in rice, but its function and structure have not been reported. Here, we report the 2.0 ? crystal structure of the kinase domain of CKI-like protein from rice. Although the structure adopts the typical bi-lobal kinase architecture, the length and orientation of the glycine-rich ATP-binding motif are dynamic within the CKI family. A loop between α5 and α6 (the α5-α6 loop), which was previously not detected in the CKI family because of flexibility, was clearly detected in our structure. In addition, we identified a lipase as a substrate of CKI-like protein from rice. Phosphorylation of the lipase dramatically reduced its catalytic activity, suggesting that CKI may play a role in the regulation of lipase activity.     

The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase.

An S-receptor kinase (SRK) cDNA, SRK-910, from the active S-locus in a self-incompatible Brassica napus W1 line has been isolated and characterized. The SRK-910 gene is predominantly expressed in pistils and segregates with the W1 self-incompatibility phenotype in an F2 population derived from a cross between the self-incompatible W1 line and a self-compatible Westar line. Analysis of the predicted amino acid sequence demonstrated that the extracellular receptor domain is highly homologous to S-locus glycoproteins, whereas the cytoplasmic kinase domain contains conserved amino acids present in serine/threonine kinases. An SRK-910 kinase protein fusion was produced in Escherichia coli and found to contain kinase activity. Phosphoamino acid analysis confirmed that only serine and threonine residues were phosphorylated. Thus, the SRK-910 gene encodes a functional serine/threonine receptor kinase.     

A family of K+ channel ancillary subunits regulate taste sensitivity in Caenorhabditis elegans

We have identified a family of ancillary subunits of K(+) channels in Caenorhabditis elegans. MPS-1 and its related members MPS-2, MPS-3, and MPS-4 are detected in the nervous system of the nematode. Electrophysiological analysis in ASE neurons and mammalian cells and epigenetic inactivation by double-stranded RNA interference (RNAi) in vivo show that each MPS can associate with and functionally endow the voltage-gated K(+) channel KVS-1. In the chemosensory neuron ADF, three different MPS subunits combine with KVS-1 to form both binary (MPS-1.KVS-1) and ternary (MPS-2.MPS-3.KVS-1) complexes. RNAi of mps-2, mps-3, or both, enhance the taste of the animal for sodium without altering the susceptibility to other attractants. When sodium is introduced in the test plate as background or as antagonist attractant, the nematode loses the ability to recognize a second attractant. Thus, it appears that the chemosensory apparatus of C. elegans uses sensory thresholds and that a voltage-gated K(+) channel is specifically required for this mechanism.     

Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase.

Epidermal growth factor stimulates the activity of several cytosolic serine/threonine protein kinases in quiescent Swiss 3T3 cells. Two of these, which use myelin basic protein (MBP) as substrate, act as kinase kinases in that they are able to activate a separate peptide kinase activity in vitro by a mechanism involving protein phosphorylation. In this study, we have identified two activities from extracts of epidermal growth factor-treated cells that stimulate an ATP-dependent activation of both of the MBP kinases, derived in their inactive precursor forms from extracts of untreated cells. The resulting MBP kinase activities are stable to further purification and can be inactivated with either tyrosine or serine/threonine protein phosphatases and then reactivated to their original levels of activity. Thus, we propose that the in vitro activation involves protein phosphorylation, stimulated by the action of novel MBP kinase activating factors that represent intermediate components in a growth factor-stimulated kinase cascade.     

Specific phosphorylation of threonine by the Dictyostelium myosin II heavy chain kinase family

Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine.     

Structure-function similarities between a plant receptor-like kinase and the human interleukin-1 receptor-associated kinase-4

Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.     

Dimerization-induced folding of MST1 SARAH and the influence of the intrinsically unstructured inhibitory domain: low thermodynamic stability of monomer

The serine/threonine mammalian sterile 20-like kinase (MST1) is involved in promotion of caspase-dependent and independent apoptosis. Phosphorylation and oligomerization are required for its activation. The oligomerization domain, denoted as SARAH domain, forms an antiparallel coiled coil dimer, and it is important for both MST1 autophosphorylation and interactions with other proteins like the Rassf proteins containing also a SARAH domain. Here we show that the monomeric state of SARAH is thermodynamically unstable and that homodimerization is coupled with folding. Moreover, the influence of the inhibitory domain on SARAH stability and affinity is addressed. By investigating the thermal denaturation using differential scanning calorimetry and circular dichroism, we have found that the SARAH domain dissociates and unfolds cooperatively, without a stable intermediate monomeric state. Combining the data with information from isothermal titration calorimetry, a low thermodynamic stability of the monomeric species is obtained. Thus, it is proposed that the transition from MST1 SARAH homodimer to some specific heterodimer implies a non-native monomer intermediate. The inhibitory domain is found to be highly flexible and intrinsically unfolded, not only in isolation but also in the dimeric state of the inhibitory-SARAH construct. The existence of two caspase recognition motifs within the inhibitory domain suggests that its structural flexibility might be important for activation of MST1 during apoptosis. Moreover, the inhibitory domain increases the thermodynamic stability of the SARAH dimer and the homodimer affinity, while having almost no effect on the SARAH domain in the monomeric state. These results emphasize the importance of flexibility and binding-induced folding for specificity, affinity, and the capacity to switch from one state to another.