A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs)

A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)+ RNAs of tobacco cells in suspension culture. The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs. The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues. These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase. Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2. The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms. Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.     

Regulation of RasGRP via a phorbol ester-responsive C1 domain

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.     

Receptor-like protein kinase genes of Arabidopsis thaliana

The isolation of a maize cDNA clone that encodes a membrane spanning protein kinase related to the self-incompatibility glycoproteins (SLG) of Brassica and structurally similar to the growth factor receptor tyrosine kinases has recently been reported. Three distinct receptor-like protein kinase (RLK) cDNA clones from Arabidopsis thaliana have now been identified. Two of the Arabidopsis RLK genes encode SLG-related protein kinases but have different patterns of expression: one is expressed predominantly in rosettes while the other is expressed primarily in roots. The third RLK gene contains an extracellular domain that consists of 21 leucine-rich repeats that are analogous to the leucine-rich repeats found in proteins from humans, flies and yeast. The Arabidopsis leucine-rich gene is expressed at equivalent levels in roots and rosettes. These results show that there are several genes in higher plants that encode members of the receptor protein kinase superfamily. The structural diversity and differential expression of these genes suggest that each plays a distinct and possibly important role in cellular signaling in plants.     

Presence of a non-NMDA glutamate receptor subtype in the sympathetic nervous system of neonatal swine

Calcium-dependent protein kinases (CDPKs) in plants are characterized by a four-domain structure including conserved sequences in the catalytic domain, and in the C-terminal calmodulin-like domain. Based on this conservation we have PCR-amplified and isolated a potato cDNA clone (StCPK1) from a library representing an early stage of tuber development. DNA sequence analysis revealed that in the catalytic domain, StCPK1 shares more homology with CDPK-related kinases than with CDPKs; however, like CDPKs, it possesses canonical EF-hands at the calmodulin-like 3' end. StCPK1 exists in a few copies in the potato genome and is abundantly expressed in the sepals of mature flowers. Floral expression of genes homologous to StCPK1 appears to be widespread in the family Solanaceae.     

Plant calcium-dependent protein kinase-related kinases (CRKs) do not require calcium for their activities

In plants, calcium-dependent protein kinases (CDPKs) make up a large family that is characterized by a C-terminal calmodulin(CaM)-like domain. Recently, a novel carrot cDNA clone encoding an atypical CDPK, which has a significantly degenerate sequence in the CaM-like domain, was found and named CDPK-related protein kinase (CRK) [Lindzen, E. and Choi, J.H. (1995) Plant Mol. Biol. 28, 785-797]. We obtained two different cDNA clones from maize which encode CRKs. For the first enzymatic characterization of CRK, a maize cDNA clone was expressed in E. coli. The recombinant protein efficiently phosphorylated casein, a conventional protein substrate. Notably, in this in vitro phosphorylation assay, the kinase activity did not require calcium as an activator. Thus, CRKs were suggested to be novel calcium-independent protein kinases having a degenerate CaM domain, the function of which remains to be elucidated.     

Value of posterior cruciate ligament index in the diagnosis of anterior cruciate ligament injuries

Several EF-hand recoverin mutants were obtained and their abilities to bind to photoreceptor membranes and to inhibit rhodopsin kinase were determined. The mutants with the 'spoiled' 2nd, 3rd or (2nd+3rd) EF-hand structures did not act upon the kinase activity in the microM range of Ca2+ concentrations. Mutations of the 4th EF hand, which 'repaired' its Ca2+-binding activity, resulted in recoverin with three 'working' Ca2+-binding sites. The latter mutant inhibited rhodopsin kinase even more effectively than the wild-type recoverin, containing two working Ca2+-binding structures.     

Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase

Screening of a yeast two-hybrid library for proteins that interact with the kinase domain of an S-locus receptor kinase (SRK) resulted in the isolation of a plant protein called ARC1 (Arm Repeat Containing). This interaction was mediated by the C-terminal region of ARC1 in which five arm repeat units were identified. Using the yeast two-hybrid system and in vitro binding assays, ARC1 was found to interact specifically with the kinase domains from SRK-910 and SRK-A14 but failed to interact with kinase domains from two different Arabidopsis receptor-like kinases. In addition, treatment with a protein phosphatase or the use of a kinase-inactive mutant reduced or abolished the binding of ARC1 to the SRK-910 kinase domain, indicating that the interaction was phosphorylation dependent. Lastly, RNA blot analysis revealed that the expression of ARC1 is restricted to the stigma, the site of the self-incompatibility response.     

Crystal structure of a pair of follistatin-like and EF-hand calcium-binding domains in BM-40

BM-40 (also known as SPARC or osteonectin) is an anti-adhesive secreted glycoprotein involved in tissue remodelling. Apart from an acidic N-terminal segment, BM-40 consists of a follistatin-like (FS) domain and an EF-hand calcium-binding (EC) domain. Here we report the crystal structure at 3.1 A resolution of the FS-EC domain pair of human BM-40. The two distinct domains interact through a small interface that involves the EF-hand pair of the EC domain. Residues implicated in cell binding, inhibition of cell spreading and disassembly of focal adhesions cluster on one face of BM-40, opposite the binding epitope for collagens and the N-linked carbohydrate. The elongated FS domain is structurally related to serine protease inhibitors of the Kazal family. Notable differences are an insertion into the inhibitory loop in BM-40 and a protruding N-terminal beta-hairpin with striking similarities to epidermal growth factor. This hairpin is likely to act as a rigid spacer in proteins containing tandemly repeated FS domains, such as follistatin and agrin, and forms the heparin-binding site in follistatin.     

The amino-terminal region of Tyk2 sustains the level of interferon alpha receptor 1, a component of the interferon alpha/beta receptor

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-alpha (IFN-alpha) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-alpha. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-alpha receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficient in vitro IFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-alpha. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.     

Genetic evidence for Pak1 autoinhibition and its release by Cdc42

Pak1 protein kinase of Schizosaccharomyces pombe, a member of the p21-GTPase-activated protein kinase (PAK) family, participates in signaling pathways including sexual differentiation and morphogenesis. The regulatory domain of PAK proteins is thought to inhibit the kinase catalytic domain, as truncation of this region renders kinases more active. Here we report the detection in the two-hybrid system of the interaction between Pak1 regulatory domain and the kinase catalytic domain. Pak1 catalytic domain binds to the same highly conserved region on the regulatory domain that binds Cdc42, a GTPase protein capable of activating Pak1. Two-hybrid, mutant, and genetic analyses indicated that this intramolecular interaction rendered the kinase in a closed and inactive configuration. We show that Cdc42 can induce an open configuration of Pak1. We propose that Cdc42 interaction disrupts the intramolecular interactions of Pak1, thereby releasing the kinase from autoinhibition.     

Sequence and context dependence of EF-hand loop dynamics. An 15N relaxation study of a calcium-binding site mutant of calbindin D9k

The influence of amino acid sequence and structural context on the backbone dynamics of EF-hand calcium-binding loops was investigated using 15N spin relaxation measurements on the calcium-free state of the calbindin D9k mutant (A14D+A15Delta+P20Delta+N21G+P43M), in which the N-terminal pseudo-EF-hand loop, characteristic of S100 proteins, was engineered so as to conform with the C-terminal consensus EF-hand loop. The results were compared to a previous study of the apo state of the wild-type-like P43G calbindin D9k mutant. In the helical regions, the agreement with the P43G data is excellent, indicating that the structure and dynamics of the protein core are unaffected by the substitutions in the N-terminal loop. In the calcium-binding loops, the flexibility is drastically decreased compared to P43G, with the modified N-terminal loop showing a motional restriction comparable to that of the surrounding helixes. As in P43G, the motions in the C-terminal loop are less restricted than in the N-terminal loop. Differences in key hydrogen-bonding interactions correlate well with differences in dynamics and offer insights into the relationship between structure and dynamics of these EF-hand loops. It appears that the entire N-terminal EF-hand is built to form a rigid structure that allows calcium binding with only minor rearrangements and that the structural and dynamical properties of the entire EF-hand--rather than the loop sequence per se--is the major determinant of loop flexibility in this system.     

Characterization of an Arabidopsis thaliana gene that defines a new class of putative plant receptor kinases with an extracellular lectin-like domain

We have characterized an Arabidopsis receptor-like serine/threonine kinase gene, Ath.lecRK1 (Arabidopsis thaliana lectin-receptor kinase), defining a new and putatively important class of plant receptor kinases. Structural features of the predicted polypeptide include an amino-terminal membrane-targeting signal sequence, a legume lectin-like extracellular domain, a single membrane-spanning domain, and a characteristic serine/threonine protein kinase domain. A recombinant protein containing the kinase domain can be autophosphorylated on a serine residue. Ath.lecRK1 is a member of a gene family of at least two closely related genes. Northern blot analysis indicates that the Ath.lecRK1 gene is weakly expressed in a variety of organs and is regulated in Arabidopsis cell suspension cultures according to the growth phase of cells. The role this new class of plant receptor kinase could play is discussed with regard to the transduction of oligosaccharide and plant hormone signals.     

The mitogen-activated protein kinase phosphatase-3 N-terminal noncatalytic region is responsible for tight substrate binding and enzymatic specificity

We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactivation of the extracellular signal-regulated kinase (ERK) class of mitogen-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigues, N., Boschert, U., Camps, M., Gillieron, C., Davies, K., Ashworth, A., and Arkinstall, S. (1996) J. Biol. Chem. 271, 27205-27208). We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction with either c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) or p38 MAP kinases was detected. Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3DeltaC) binds both ERK1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3DeltaN) fails to precipitate either of these MAP kinases. A chimera consisting of the N-terminal half of MKP-3 with the C-terminal catalytic core of M3-6 also bound tightly to ERK1 but not to JNK3/SAPKbeta. Consistent with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3DeltaN than full-length MKP-3 is required to inhibit ERK2 activity. In contrast, both MKP-3DeltaN and full-length MKP-3 inactivate JNK/SAPK and p38 MAP kinases at similarly high concentrations. Also, a chimera of the M3-6 N terminus with the MKP-3 catalytic core which fails to bind ERK elicits non selective inactivation of ERK1 and JNK3/SAPKbeta. Together, these observations suggest that the physiological specificity of MKP-3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.     

A novel chloride channel in Vicia faba guard cell vacuoles activated by the serine/threonine kinase, CDPK

Calcium-Dependent Protein Kinases (CDPKs) in higher plants contain a C-terminal calmodulin-like regulatory domain. Little is known regarding physiological CDPK targets. Both kinase activity and multiple Ca2+-dependent signaling pathways have been implicated in the control of stomatal guard cell movements. To determine whether CDPK or other protein kinases could have a role in guard cell signaling, purified and recombinant kinases were applied to Vicia faba guard cell vacuoles during patch-clamp experiments. CDPK activated novel vacuolar chloride (VCL) and malate conductances in guard cells. Activation was dependent on both Ca2+ and ATP. Furthermore, VCL activation occurred in the absence of Ca2+ using a Ca2+-independent, constitutively active, CDPK* mutant. Protein kinase A showed weaker activation (22% as compared with CDPK). Current reversals in whole vacuole recordings shifted with the Nernst potential for Cl-and vanished in glutamate. Single channel recordings showed a CDPK-activated 34 +/- 5 pS Cl- channel. VCL channels were activated at physiological potentials enabling Cl- uptake into vacuoles. VCL channels may provide a previously unidentified, but necessary, pathway for anion uptake into vacuoles required for stomatal opening. CDPK-activated VCL currents were also observed in red beet vacuoles suggesting that these channels may provide a more general mechanism for kinase-dependent anion uptake.     

The Cbl phosphotyrosine-binding domain selects a D(N/D)XpY motif and binds to the Tyr292 negative regulatory phosphorylation site of ZAP-70

The Cbl protooncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases through currently undefined mechanisms. Therefore, determining how Cbl physically interacts with tyrosine kinases is of substantial interest. We recently identified a phosphotyrosine binding (PTB) domain residing within the N-terminal transforming region of Cbl (Cbl-N), which mediated direct binding to ZAP-70 tyrosine kinase. Here, we have screened a degenerate phosphopeptide library and show that the Cbl-PTB domain selects a D(N/D)XpY motif, reminiscent of but distinct from the NPXpY motif recognized by the PTB domains of Shc and IRS-1/2. A phosphopeptide predicted by this motif and corresponding to the in vivo negative regulatory phosphorylation site of ZAP-70 (Tyr(P)292) specifically inhibited binding of ZAP-70 to Cbl-N. A ZAP-70/Y292F mutant failed to bind to Cbl-N, whereas a D290A mutant resulted in a 64% decrease in binding, confirming the importance of the Tyr(P) and Y-2 residues in Cbl-PTB domain recognition. Finally the ZAP-70/Y292F mutant also failed to associate with Cbl-N or full-length Cbl in vivo. These results identify a potential Cbl-PTB domain-dependent role for Cbl in the negative regulation of ZAP-70 and predict potential Cbl-PTB domain binding sites on other protein tyrosine kinases known to interact with Cbl.     

Calcium regulation of Ndr protein kinase mediated by S100 calcium-binding proteins

Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases identified as being critical for the regulation of cell division and cell morphology. The regulatory mechanisms that control Ndr activity have not been characterized previously. In this paper, we present evidence that Ndr is regulated by EF-hand calcium-binding proteins of the S100 family, in response to changes in the intracellular calcium concentration. In vitro, S100B binds directly to and activates Ndr in a Ca2+-dependent manner. Moreover, Ndr is recovered from cell lysates in anti-S100B immunoprecipitates. The region of Ndr responsible for interaction with Ca2+/S100B is a basic/hydrophobic motif within the N-terminal regulatory domain of Ndr, and activation of Ndr by Ca2+/S100B is inhibited by a synthetic peptide derived from this region. In cultured cells, Ndr is rapidly activated following treatment with Ca2+ ionophore, and this activation is dependent upon the identified Ca2+/S100B-binding domain. Finally, Ndr activity is inhibited by W-7 in melanoma cells overexpressing S100B, but is unaffected by W-7 in melanoma cells that lack S100B. These results suggest that Ndr is regulated at least in part by changes in the intracellular calcium concentration, through binding of S100 proteins to its N-terminal regulatory domain.     

Cla4p, a Saccharomyces cerevisiae Cdc42p-activated kinase involved in cytokinesis, is activated at mitosis

Yeasts have three functionally redundant G1 cyclins required for cell cycle progression through G1. Mutations in GIN4 and CLA4 were isolated in a screen for mutants that are inviable with deletions in the G1 cyclins CLN1 and CLN2. cln1 cln2 cla4 and cln1 cln2 gin4 cells arrest with a cytokinesis defect; this defect was efficiently rescued by CLN1 or CLN2 expression. GIN4 encodes a protein with strong homology to the Snflp serine/threonine kinase. Cla4p is homologous to mammalian p21-activated kinases (PAKs) (kinases activated by the rho-class GTPase Rac or Cdc42). We developed a kinase assay for Cla4p. Cla4p kinase was activated in vivo by the GTP-bound form of Cdc42p. The specific activity of Cla4p was cell cycle regulated, peaking near mitosis. Deletion of the Cla4p pleckstrin domain diminished kinase activity nearly threefold and eliminated in vivo activity. Deletion of the Cla4p Cdc42-binding domain increased kinase activity nearly threefold, but the mutant only weakly rescued cla4 function in vivo. This suggests that kinase activity alone is not sufficient for full function in vivo. Deletion of the Cdc42-binding domain also altered the cell cycle regulation of kinase activity. Instead of peaking at mitosis, the mutant kinase activity exhibited reduced cell cycle regulation and peaked at the G1/S border. Cla4p kinase activity was not reduced by mutational inactivation of gin4, suggesting that Gin4p may be downstream or parallel to Cla4p in the regulation of cytokinesis.     

Complexes between STE5 and components of the pheromone-responsive mitogen-activated protein kinase module

We present genetic evidence for complex formation of STE5 and the STE11, STE7, and FUS3 protein kinases, the pheromone-responsive mitogen-activated protein kinase module of Saccharomyces cerevisiae. Interaction between STE5 and STE11 is not dependent on STE7, and interaction between STE5 and STE7 does not require STE11. The N-terminal regulatory domain of STE11 is both necessary and sufficient for interaction with STE5. Interaction between STE7 and STE11 is bridged by STE5, suggesting the formation of a multiprotein complex. We also demonstrate biochemical interaction between STE5 and STE11 by using a combination of bacterially expressed fusion proteins and extracts prepared from yeast. Our results suggest that STE5 is a scaffolding protein that facilitates interactions between components of the pheromone-responsive mitogen-activated protein kinase module. We further propose that such scaffolding proteins serve to inhibit cross-talk between functionally unrelated mitogen-activated protein kinase modules within the same cell.     

The 2.35 A crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regulatory interactions

The Src protein tyrosine kinase plays a critical role in a variety of signal transduction pathways. Strict regulation of its activity is necessary for proper signalling. We present here the crystal structure of chicken Src which is phosphorylated at Tyr527 and represents its least active form. Our structure, similar to the recently reported human Hck and Src structures, contains the SH3, SH2 and the kinase domains and the C-terminal regulatory tail but not the N-terminal unique domain. The SH3 domain uses its hydrophobic surface to coordinate the SH2-kinase linker such that residues Gln251 and Leu255 specifically interact with side chains in the beta2-beta3 and the alphaC-beta4 loops of the N-terminal lobe opposite of the kinase active site. This position of the SH3 domain and the coordination of the SH2-kinase linker also optimally places the SH2 domain such that the phosphorylated Tyr527 in the C-terminal tail interacts with the SH2 binding pocket. Analogous to Cdk2 kinase, the position of the Src alphaC-helix in the N-terminal lobe is swung out disrupting the position of the active site residues. Superposition of other protein kinases including human Hck and Src onto chicken Src indicate that the alphaC-helix position is affected by the relative position of the N-terminal lobe with respect to the C-terminal lobe of the kinase and that the presence of the SH3/SH2-kinase linker/N-terminal lobe interactions restricts the kinase lobes and alphaC-helix access to the active conformation. These superpositions also suggest that the highly conserved alphaC-beta4 loop restricts the conformational freedom of the N-terminal lobe by anchoring it to the C-terminal lobe. Finally, based on sequence alignments and conservation of hydrophobic residues in the Src SH2-kinase linker as well as in the alphaC-beta4 and beta2-beta3 loops, we propose that the Src-related kinases, Abl, Btk and Csk, share the same quaternary structure.