Identification and characterization of PKNbeta, a novel isoform of protein kinase PKN: expression and arachidonic acid dependency are different from those of PKNalpha.

The cDNA clone encoding a novel isoform of protein kinase PKN, termed PKNbeta, was isolated from a HeLa cDNA library. PKNbeta had high sequence homology with PKNalpha, originally isolated PKN, especially in the repeats of charged amino acid-rich region with leucine-zipper like sequences (CZ region/HR1), in the carboxyl-terminal catalytic domain, and in approximately 130 amino acid stretch (D region/HR2), located between CZ region/HR1 and the catalytic domain. However, the amino acid sequence of PKNbeta differed from that of PKNalpha in the region immediately amino-terminal to the catalytic domain, which contained two distinct proline-rich sequences consistent with the class II consensus sequence, PXXPXR, for binding to SH3 domain. Distribution of PKNbeta differed from that of PKNalpha in the following two respects: (1) Northern blotting indicated that PKNbeta mRNA could not be detected in human adult tissues, but was expressed abundantly in human cancer cell lines; (2) immunochemical analysis indicated that PKNbeta localized in nucleus and perinuclear Golgi apparatus, and was almost absent in cytoplasmic region in NIH3T3 cells. Recombinant PKNbeta expressed in COS7 cells displayed autophosphorylation and peptide kinase activity, but was found to be significantly less responsive to arachidonic acid than PKNalpha. The identification of this novel isoform underscores the diversity of PKN signaling pathway.     

Characterization of elk, a brain-specific receptor tyrosine kinase.

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extracellular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system.     

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.     

Identification of rat cDNA encoding hck tyrosine kinase from megakaryocytes.

A rat homologue of hck tyrosine kinase cDNA from a rat megakaryocyte library contains 1911 nucleotides with an open reading frame encoding 503 amino acids. The rat hck had distinct amino acid residues from the mouse homologue exhibiting 97.6% identity. The sequence contains the SH2 and SH3 regions that interact with cytoplasmic signaling proteins, the kinase domain including the nucleotide binding site and the autophosphorylation site, and the C-terminal Tyr-499 known as a negative regulator.     

Identification of the autophosphorylation sites in rhodopsin kinase.

Rhodopsin kinase (RK) is a second-messenger-independent protein kinase that is involved in deactivation of photolyzed rhodopsin (Rho*). We have developed a significantly improved method for isolation of RK based on the specific interactions of phosphorylated forms of the enzyme with heparin-Sepharose. Conversion of the dephosphorylated form of RK to the fully phosphorylated enzyme leads to specific elution of the kinase from the resin. Limited proteolysis of RK with endoproteinase Asp-N removes the phosphorylation sites. Peptides containing the autophosphorylation sites were isolated by reverse-phase high performance liquid chromatography and analyzed by Edman degradation and tandem mass spectrometry. The derived amino acid sequence of the peptide containing the major autophosphorylation site yielded the following sequence: DVGAFS488T489VKGVAFEK, where Ser488 and Thr489 are phosphorylated. Additionally, a minor autophosphorylation site was identified at Ser21. A 15-residue peptide (DVGAFSTVKGVAFEK) encompassing the major autophosphorylation site was synthesized and used for phosphorylation and inhibition studies. In contrast to many other protein kinases, the low catalytic activity of RK toward its autophosphorylation site peptide and the poor inhibitory properties of this peptide suggest unique properties of this member of the family of G protein-coupled receptor kinases.     

Direct binding of syndecan-4 cytoplasmic domain to the catalytic domain of protein kinase C alpha (PKC alpha) increases focal adhesion localization of PKC alpha

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that acts as a coreceptor with integrins in focal adhesion formation. The central region of syndecan-4 cytoplasmic domain (4V; LGKKPIYKK) binds phosphatidylinositol 4,5-bisphosphate, and together they regulate protein kinase C alpha (PKC alpha) activity. Syndecan 4V peptide directly potentiates PKC alpha activity, leading to "superactivation" of the enzyme, apparently through an interaction with its catalytic domain. We now have performed yeast two-hybrid and in vitro binding assays to determine the interaction sites between 4V and PKC alpha. Full-length PKC alpha weakly interacted with 4V by yeast two-hybrid assays, but PKC alpha constructs that lack the pseudosubstrate region or constructs of the whole catalytic domain interacted more strongly. A mutated 4V sequence (4V(YF): LGKKPIFKK) did not interact with PKC alpha, indicating that tyrosine 192 in the syndecan-4 cytoplasmic domain might be critical for this interaction. Further assays identified a novel interaction site in the C terminus of the catalytic domain of PKC alpha (amino acid sequence 513-672). This encompasses the autophosphorylation sites, which are implicated in activation and stability. Yeast two-hybrid data were confirmed by in vitro binding and coimmunoprecipitation assays. The interaction of syndecan-4 with PKC alpha appears unique since PKC delta and epsilon did not interact with 4V in yeast two-hybrid assays or coimmunoprecipitate with syndecan-4. Finally, overexpression of syndecan-4 in rat embryo fibroblast cells, but not expression of the YF mutant, increased PKC alpha localization to focal adhesions. The data support a mechanism where syndecan-4 binds PKC alpha and localizes it to focal adhesions, whose assembly may be regulated by the kinase.     

Autophosphorylation is required for high kinase activity and efficient transformation ability of proteins encoded by host range alleles of v-src.

pp60v-src is a nonreceptor protein tyrosine kinase that can transform both chicken and rodent fibroblasts. The src homology 2 (SH2) domain of this protein serves a critical role in the regulation of protein tyrosine kinase activity. The host range proteins pp60v-src-L, which contains a deletion of a highly conserved residue (Phe-172) in the SH2 domain, and pp60v-src-PPP, which contains a change from a Leu to a Phe at amino acid 186 in the SH2 domain, transform chicken but not rat cells and have slightly reduced kinase activity measured in vitro. The data presented here show that these altered proteins require autophosphorylation on Tyr-416 for high kinase activity and transforming ability. In the absence of autophosphorylation, there is a further decrease of at least threefold in in vitro kinase activity relative to the phosphorylated host range parental protein, no morphological transformation, a reduction in anchorage independent growth, and no disruption of the actin cytoskeleton. In addition, these SH2 mutations abolish the ability of the SH2 domain to bind a phosphorylated peptide that corresponds to the autophosphorylation site of pp60src. Thus, like mutant alleles of c-src encoding transformation competent proteins, and unlike v-src, transformation by pp60v-src-F172 delta and pp60v-src-L186F is dependent on phosphorylation of Y-416 for high kinase activity and transformation ability. The dependence of transformation on phosphotyrosine is not a reflection of an intramolecular interaction between the autophosphorylation site and the SH2 domains since purified SH2 domains are incapable of binding phosphorylated autophosphorylation site peptides in vitro.     

Cloning and Analyses of a Dual Specific Serine􊄯Thronine Protein Kinase Gene with High Conservative and Constitutive Expression in Oryza (OsSTK)

A cytoplasmic serine􊄯thronine protein kinase gene (OsSTK), had been cloned from Oryza genus. It was found high conservative and constitutive expression in Oryza. OsSTK gene had two exons, separated by 114 bp short intron. The open reading frame of OsSTK gene that predicted encoded a 419 amino acids protein . The amino acid sequence of OsSTK had low identities ( less than 53% ) with any other known protein kinase . The phylogenetic tree based on the partial DNA sequences of OsSTK from different species and types of wild rice and cultivated rice, was close to the taxation system ofrice . Interestingly , OsSTK had a serine, including basic amino acids and charged amino acids abundant polypeptide with a “GDGDGDGDG”sequence at N- terminal that had not been found in any other genes. OsSTK may play dual specificity that phosphorylates both serine􊄯thronine and tyrosone, because the amino acids module of VIb , VIII and XI catalytic domain have both the serine􊄯thronine and tyrosine kinase characters .     

A novel transmembrane serine/threonine protein kinase gene from a rifamycin SV-producing amycolatopsis mediterranei U32.

Genomic DNA sequencing in the vicinity of methylmalonyl-CoA mutase gene (mutAB) from a rifamycin SV-producing Amycolatopsis mediterranei U32 allowed us to clone, sequence, and identify a gene encoding a novel serine/threonine protein kinase (amk). The sequence contains a complete ORF of 1821 base pairs encoding a predicted protein of 606 amino acids in length. The N-terminal domain of the protein shows significant homology to the catalytic domain of other protein kinases from both prokaryotic and eukaryotic sources. It also contains all the structural features that are highly conserved in active protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP-binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein kinase. This protein kinase gene was expressed in Escherichia coli and was shown to have the ability of autophosphorylation. The autophosphorylated site was found to be the threonine at position 164 by labeled phosphoamino acid analysis and site-directed mutagenesis. The C-terminal half of protein kinase was found to contain strong transmembrane structures by PhoA fusion protein analysis, suggesting that Amk protein kinase is a transmembrane protein. A Southern hybridization experiment showed that this type of protein kinase is distributed ubiquitously and might play significant physiological roles in the various species of streptomycetes. However, overexpression of amk gene in Streptomyces cinnamonensis showed no effect on methylmalonyl-CoA mutase activity, monensin production and the hyphae morphology. Although its biological role is still unknown, Amk protein kinase is the first transmembrane serine/threonine protein kinase described for genus Amycolatopsis.     

稻属中一个高度保守和组成型表达酪氨酸／丝氨酸－酸酸双特性蛋白激酶基因的克隆与分析

从水稻中克隆了一个在稻属植物中高度保守和组成型表达的丝氨酸／苏氨酸蛋白激酶基因（OsSTK）。该基因包含两个外显子和一个114bp的小内含子序列,预测编码一个419个氨基酸的蛋白质。该基因推导的氨基酸序列与其它已知序列的一致性均低于52％。利用从不同种和类型的野生稻克隆的部分该基因序列构建的系统树与野生稻的分类和进化关系相一致。OSPKN-端拥有一段富含丝氨酸、碱性氨基酸和带电荷氨基酸的特异性导肽序列,其中包含“GDGDGDGDG”短重复序列。由于该基因蛋白激酶结构域中的VIb,VIII和XI亚结构域中同时具有酪氨酸蛋白激酶和丝氨酸／苏氨酸蛋白激酶的特性,推测该基因可能同时具有催化酪氨酸和丝氨酸、苏氨酸磷酸化的双重功能。     

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.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">OsBISERK1</Emphasis>, a gene encoding a leucine-rich repeat receptor-like kinase,during disease resistance responses in rice

A rice gene, OsBISERK1, encoding a protein belonging to SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) type of leucine-rich repeat receptor-like kinases (LRR-RLKs) was identified. The OsBISERK1 encodes a 624 aa protein with high level of identity to known plant SERKs. OsBISERK1 contains a hydrophobic signal peptide, a leucine zipper, and five leucine-rich repeat motifs in the extracellular domain; the cytoplasmic region carries a proline-rich region and a single transmembrane domain, as well as a conserved intracellular serine/threonine protein kinase domain. OsBISERK1 has a low level of basal expression in leaf tissue. However, expression of OsBISERK1 was induced by treatment with benzothiadiazole (BTH), which is capable of inducing disease resistance in rice, and also up-regulated after inoculation with Magnaporthe grisea in BTH-treated rice seedlings and during incompatible interaction between a blast-resistant rice genotype and M. grisea. The results suggest that OsBISERK1 may be involved in disease resistance responses in rice.     

Cloning and biochemical properties of CDPK gene OsCDPK14 from rice

A rice CDPK gene, OsCDPK14 (AY144497), was cloned from developing caryopses of rice (Oryza sativa cv. Zhonghua 15). Its cDNA sequence (1922 bp) contains an ORF encoding a 514 amino acids protein (56.7kD, pl 5.18). OsCDPK14 shows the typical structural features of the CDPK family, including a conserved catalytic Ser/Thr kinase domain, an autoinhibitory domain and a CaM-like domain with four putative Ca2+-binding EF hands. Subcellular targeting indicated that OsCDPK14 was located in the cytoplasm, probably due to the absence of myristoylation and palmitoylation motifs. OsCDPK14 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using Syntide-2, a synthetic peptide. Kinase activity was shown to be Ca2+-dependent, and this activation was strongly enhanced by Mn2+ and inhibited by W7 in vitro. These results provide significant insights into the regulation and biochemical properties of OsCDPK14, suggesting OsCDPK14 may be a signal factor of cytoplasm in rice plant.     

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.     

Vascular endothelial growth factor receptor KDR tyrosine kinase activity is increased by autophosphorylation of two activation loop tyrosine residues

Vascular endothelial growth factor is an important physiological regulator of angiogenesis. The function of this endothelial cell selective growth factor is mediated by two homologous tyrosine kinase receptors, fms-like tyrosine kinase 1 (Flt-1) and kinase domain receptor (KDR). Although the functional consequence of vascular endothelial growth factor binding to the Flt-1 receptor is not fully understood, it is well established that mitogenic signaling is mediated by KDR. Upon sequencing several independent cDNA clones spanning the cytoplasmic region of human KDR, we identified and confirmed the identity of a functionally required valine at position 848 in the ATP binding site, rather than the previously reported glutamic acid residue, which corresponds to an inactive tyrosine kinase. The cytoplasmic domain of recombinant native KDR, expressed as a glutathione S-transferase fusion protein, can undergo autophosphorylation in the presence of ATP. In addition, the kinase activity can be substantially increased by autophosphorylation at physiologic ATP concentrations. Mutation analysis indicates that both tyrosine residues 1054 and 1059 are required for activation, which is a consequence of an increased affinity for both ATP and the peptide substrate and has no effect on kcat, the intrinsic catalytic activity of the enzyme. KDR kinase catalyzes phosphotransfer by formation of a ternary complex with ATP and the peptide substrate. We demonstrate that tyrosine kinase antagonists can preferentially inhibit either the unactivated or activated form of the enzyme.     

A truncated protein kinase domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) expressed in Escherichia coli.

The amino-terminal domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) contains a serine/threonine-specific protein kinase that has characteristics of a growth factor receptor (Chung, T. D., Wymer, J. P., Smith, C. C., Kulka, M., and Aurelian, L. (1989) J. Virol. 63, 3389-3398; Chung, T. D., Wymer, J. P., Kulka, M. Smith, C. C., and Aurelian, L. (1990) Virology 179, 168-178). To characterize this protein kinase (PK) domain further we constructed a bacterial expression vector (pJL11) containing DNA sequences encoding ICP10 amino acid residues 1-445. Bacteria containing pJL11 were induced to express a 29-kDa protein (designated pp29la1) that represents a truncated portion of the ICP10-PK domain (includes PK catalytic motifs I-V) as demonstrated by immunoprecipitation with antibodies that recognize different antigenic domains, competition studies with extracts of ICP10-positive eukaryotic cells, and peptide mapping.pp29la1 has autophosphorylating and transphosphorylating activity for calmodulin. The enzyme is activated by Mn2+ but not by Mg2+ ions, and autophosphorylation is inhibited by histone. It differs from the authentic ICP10-PK in that phosphorylation is specific only for threonine.     

Active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II. Purification, characterization, and structural analysis.

We report the purification and characterization of an active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II, derived from autophosphorylation and subsequent limited chymotryptic digestion of the purified rat forebrain soluble kinase. The purified fragment was completely Ca2+/calmodulin-independent, existed as a monomer, and phosphorylated synapsin I at the same sites as does the native form of Ca2+/calmodulin-dependent protein kinase II. Kinetic studies with the purified fragment revealed a more than 10-fold increase in Vmax and a 50% decrease in Km for synthetic peptide substrates, compared with native Ca2+/calmodulin-dependent protein kinase II. No 32P-labeled autophosphorylated residues were detected in the purified active fragment, indicating that the autophosphorylation sites were not contained within this fragment. Comparative studies of this active fragment (30 kDa) and its inactive counterpart (32-kDa fragment) revealed certain structural details of both fragments. Calmodulin-overlay study, immunoblot analysis, and direct amino acid sequencing suggest that both fragments contain the entire NH2-terminal catalytic domain and were generated by distinct cleavage within the regulatory domain. The putative cleavage sites for both fragments are discussed.     

稻属中一个高度保守和组成型表达酪氨酸丝氨酸-酸酸双特性蛋白激酶基因的克隆与分析

从水稻中克隆了一个在稻属植物中高度保守和组成型表达的丝氨酸&#1089839;苏氨酸蛋白激酶基因(OsSTK)。该基因包含两个外显子和一个114 bp 的小内含子序列, 预测编码一个419 个氨基酸的蛋白质。该基因推导的氨基酸序列与其它已知序列的一致性均低于52%。利用从不同种和类型的野生稻克隆的部分该基因序列构建的系统树与野生稻的分类和进化关系相一致。OSPK N-端拥有一段富含丝氨酸、碱性氨基酸和带电荷氨基酸的特异性导肽序列, 其中包含“GDGDGDGDG”短重复序列。由于该基因蛋白激酶结构域中的VIb , VIII 和XI 亚结构域中同时具有酪氨酸蛋白激酶和丝氨酸&#1089839;苏氨酸蛋白激酶的特性, 推测该基因可能同时具有催化酪氨酸和丝氨酸、苏氨酸磷酸化的双重功能。     

SDF-2 Induction of Terminal Differentiation in Dictyostelium discoideum Is Mediated by the Membrane-Spanning Sensor Kinase DhkA

SDF-2 is a peptide released by prestalk cells during culmination that stimulates prespore cells to encapsulate. Genetic evidence indicates that the response is dependent on the dhkA gene. This gene encodes a member of the histidine kinase family of genes that functions in two-component signal transduction pathways. The sequence of the N-terminal half of DhkA predicts two hydrophobic domains separated by a 310-amino-acid loop that could bind a ligand. By inserting MYC6 epitopes into DhkA, we were able to show that the loop is extracellular while the catalytic domain is cytoplasmic. Cells expressing the MYC epitope in the extracellular domain of DhkA were found to respond only if induced with 100-fold-higher levels of SDF-2 than required to induce dhkA+ cells; however, they could be induced to sporulate by addition of antibodies specific to the MYC epitope. To examine the enzymatic activity of DhkA, we purified the catalytic domain following expression in bacteria and observed incorporation of labelled phosphate from ATP consistent with histidine autophosphorylation. Site-directed mutagenesis of histidine1395 to glutamine in the catalytic domain blocked autophosphorylation. Furthermore, genetic analyses showed that histidine1395 and the relay aspartate2075 of DhkA are both critical to its function but that another histidine kinase, DhkB, can partially compensate for the lack of DhkA activity. Sporulation is drastically reduced in double mutants lacking both DhkA and DhkB. Suppressor studies indicate that the cyclic AMP (cAMP) phosphodiesterase RegA and the cAMP-dependent protein kinase PKA act downstream of DhkA.