Novel Ca/calmodulin-dependent protein kinase expressed in actively growing mycelia of the basidiomycetous mushroom Coprinus cinereus

We isolated cDNA clones for novel protein kinases by expression screening of a cDNA library from the basidiomycetous mushroom Coprinus cinereus. One of the isolated clones was found to encode a calmodulin (CaM)-binding protein consisting of 488 amino acid residues with a predicted molecular weight of 53,906, which we designated CoPK12. The amino acid sequence of the catalytic domain of CoPK12 showed 46% identity with those of rat Ca²⁺/CaM-dependent protein kinase (CaMK) I and CaMKIV. However, a striking difference between these kinases is that the critical Thr residue in the activating phosphorylation site of CaMKI/IV is replaced by a Glu residue at the identical position in CoPK12. As predicted from its primary sequence, CoPK12 was found to behave like an activated form of CaMKI phosphorylated by an upstream CaMK kinase, indicating that CoPK12 is a unique CaMK with different properties from those of the well-characterized CaMKI, II, and IV. CoPK12 was abundantly expressed in actively growing mycelia and phosphorylated various proteins, including endogenous substrates, in the presence of Ca²⁺/CaM. Treatment of mycelia of C. cinereus with KN-93, which was found to inhibit CoPK12, resulted in a significant reduction in growth rate of mycelia. These results suggest that CoPK12 is a new type of multifunctional CaMK expressed in C. cinereus, and that it may play an important role in the mycelial growth.     

Characterization of CoPK02, a Ca2+/calmodulin-dependent protein kinase in mushroom Coprinopsis cinerea

We surveyed genome sequences from the basidiomycetous mushroom Coprinopsis cinerea and isolated a cDNA homologous to CMKA, a calmodulin-dependent protein kinase (CaMK) in Aspergillus nidulans. We designated this sequence, encoding 580 amino acids with a molecular weight of 63,987, as CoPK02. CoPK02 possessed twelve subdomains specific to protein kinases and exhibited 43, 35, 40% identity with rat CaMKI, CaMKII, CaMKIV, respectively, and 40% identity with CoPK12, one of the CaMK orthologs in C. cinerea. CoPK02 showed significant autophosphorylation activity and phosphorylated exogenous proteins in the presence of Ca²⁺/CaM. By the CaM-overlay assay we confirmed that the C-terminal sequence (Trp346-Arg358) was the calmodulin-binding site, and that the binding of Ca²⁺/CaM to CoPK02 was reduced by the autophosphorylation of CoPK02. Since CoPK02 evolved in a different clade from CoPK12, and showed different gene expression compared to that of CoPK32, which is homologous to mitogen-activated protein kinase-activated protein kinase, CoPK02 and CoPK12 might cooperatively regulate Ca²⁺-signaling in C. cinerea.     

CoPK32 is a novel stress-responsive protein kinase in the mushroom Coprinopsis cinerea

Background

In a previous study, we conducted an expression cloning screen of a cDNA library prepared from Coprinopsis cinerea mycelia using Multi-PK antibodies and detected a wide variety of Ser/Thr protein kinases. One of the isolated clones, CMZ032, was found to encode a putative Ser/Thr protein kinase designated CoPK32. In the present study, we investigated the biochemical properties and physiological significance of CoPK32.

Methods

CoPK32 was expressed in Escherichia coli, and its biochemical properties were examined. The effects of high osmotic stresses on the growth of C. cinerea and on the endogenous CoPK32 activity in mycelia were also examined.

Results

CoPK32 showed autophosphorylation activity and effectively phosphorylated exogenous protein substrates. CoPK32S, a splice variant that was 18 amino acids shorter than CoPK32, showed much lower protein kinase activity than CoPK32. The catalytic properties of CoPK32 deletion mutants suggested that the C-terminal region of CoPK32 was important for the kinase activity and recognition of substrates. CoPK32 was highly expressed in the actively growing region of the mycelial colony. When mycelia were stimulated by high osmotic stresses, endogenous CoPK32 was markedly activated and the mycelial growth was severely inhibited. The activation of CoPK32 activity by high osmotic stresses was abrogated by SB202190 or SB239063 as well-known inhibitors of p38 mitogen-activated protein kinase.

Conclusions

CoPK32 is involved in the stress response pathway in mycelia of C. cinerea in response to environmental stresses.

General significance

In C. cinerea, protein kinases such as CoPK32 play important roles in signal transduction pathways involved in stress responses.     

Chromosomal localization of the human gene for brain Ca2+/calmodulin-dependent protein kinase type IV

Cloned cDNAs have been identified as corresponding to a new brain Ca2+/calmodulin-dependent protein kinase. On the basis of structural and immunological features, we refer to this new kinase as CaM Kinase IV. Two cDNA clones were used to identify CaM Kinase IV: The downstream clone, lambda ICM-1, contains the sequence encoding the calmodulin-binding site and the second clone, lambda ICM-2, encodes a partial amino acid sequence similar to the catalytic domain of several known protein kinases. Within the calmodulin-binding site a stretch of 8 amino acids (and 9 of 10) is identical to the corresponding site in the subunits of CaM Kinase II. Southern blot analysis shows the CaM Kinase IV gene is single copy in the mouse and human genomes. Synteny analysis of Southern blot data of DNA from hamster--human hybrid cells shows that the gene is present in human chromosome 5. Hybridization of cDNA probes to metaphase spreads of human chromosomes indicates that the gene is most likely located within the region of bands q21 to q23 of chromosome 5.     

Cloning and characterization of a novel Ca2+/calmodulin-dependent protein kinase I homologue in Xenopus laevis

In order to investigate protein kinases expressed in the different developmental stages of Xenopus laevis, recently developed expression cloning was carried out. When two different expression libraries, Xenopus oocyte and Xenopus head (embryonic stage 28/30) cDNA libraries, were screened by kinase-specific monoclonal antibodies, cDNA clones for various known and novel protein serine/threonine kinases (Ser/Thr kinases) were isolated. In addition to well-characterized Ser/Thr kinases, one cDNA clone for a putative kinase was isolated from the Xenopus head library. The sequence of the open reading frame of the cDNA encoded a protein of 337 amino acid residues with a predicted molecular weight of 38,404. Since the deduced animo acid sequence of this protein was 75% identical to that of rat Ca(2+)/calmodulin-dependent protein kinase I (CaMKI), it was designated as CaMKIx. Although recombinant CaMKIx expressed in Escherichia coli showed no protein kinase activity against syntide-2, a synthetic peptide substrate, it was activated when phosphorylated by mouse Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaMKKalpha). Activated CaMKIx significantly phosphorylated various proteins including synapsin I, histones, and myelin basic protein. CaMKIx could not be detected in the early stages of embryogenesis, but was detected in late embryos of stages 37/38 and thereafter when examined by Western blotting using a specific antibody. This kinase was found to be highly expressed in adult brain and heart, and an upstream kinase that could activate CaMKIx was detected in these tissues. These results suggest that CaMKIx plays some critical role in the late stages of embryogenesis of Xenopus laevis.     

Involvement of Ca(2+)/calmodulin-dependent protein kinases in mycelial growth of the basidiomycetous mushroom, Coprinus cinereus

Although multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) are widely distributed in animal cells, the occurrence of CaM-kinases in the basidiomycetous mushroom has not previously been documented. When the extracts from various developmental stages from mycelia to the mature fruiting body of Coprinus cinereus were analyzed by Western blotting using Multi-PK antibodies, which had been generated to detect a wide variety of protein serine/threonine kinases (Ser/Thr kinases), a variety of stage-specific Ser/Thr kinases was detected. Calmodulin (CaM) overlay assay using digoxigenin-labeled CaM detected protein bands of 65 kDa, 58 kDa, 46 kDa, 42 kDa, and 38 kDa only in the presence of CaCl(2), suggesting that these bands were CaM-binding proteins. When the CaM-binding fraction was prepared from mycelial extract of C. cinereus by CaM-Sepharose and analyzed with Multi-PK antibodies, two major immunoreactive bands corresponding to 65 kDa and 46 kDa were detected. CaM-binding fraction, thus obtained, exhibited Ca(2+)/CaM-dependent protein kinase activity toward protein substrates such as histones. These CaM-kinases were found to be highly expressed in the actively growing mycelia, but not in the resting mycelial cells. Mycelial growth was enhanced by the addition of CaCl(2) in the culture media, but inhibited by the addition of EGTA or trifluoperazine, a potent CaM inhibitor. This suggested that CaM-dependent enzymes including CaM-kinases play crucial roles in mycelial growth of basidiomycete C. cinereus.     

Catalytic and regulatory domains of doublecortin kinase-1

Doublecortin kinase-1 (DCK1) is a newly described multidomain protein kinase with a sequence significantly similar to those of both CaM kinases (CaMKs) and doublecortin, the product of the gene mutated in X-linked lissencephaly/double cortex syndrome, a severe developmental disorder of the nervous system. Functional studies have revealed microtubule binding and polymerization activities of the doublecortin domain, yet little is known regarding the enzymatic properties and regulation of the kinase catalytic domain. We have identified and report here notable similarities as well as differences between the catalytic and regulatory properties of DCK1 and those of the CaMKs. Using synthetic peptide substrates modeled on synapsin I, a substrate recognition motif for DCK1 of Hyd-Arg-Arg-X-X-Ser/Thr-Hyd was derived. The similarity of this motif to that of CaMKI [Lee, J. C., Kwon, Y.-G., Lawrence, D. S., and Edelman, A. M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6413-6417] is consistent with the 59% level of amino acid sequence similarity between their catalytic domains. DCK1 catalytic activity is enhanced by mutagenic introduction of negative charge at Thr-239, a residue in a position equivalent to that of Thr-177 of CaMKI, the activation loop site for regulation by CaM kinase kinase. Unlike CaMKs, DCK1 is not directly activated by Ca(2+)-bound CaM. However, truncation of a pseudosubstrate-like sequence in the C-terminus of DCK1 results in an approximately 6-fold enhancement of activity. Thus, DCK1 demonstrates the potential to be regulated by relief of autoinhibition in response to signal(s) distinct from Ca(2+)-bound CaM and potentially by activation loop phosphorylation and to phosphorylate intracellular targets at sites similar to those recognized by CaMK pathways.     

Characterization of a novel calcium/calmodulin-dependent protein kinase from tobacco

A cDNA encoding a calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CaMK) from tobacco (Nicotiana tabacum), NtCaMK1, was isolated by protein-protein interaction-based screening of a cDNA expression library using 35S-labeled CaM as a probe. The genomic sequence is about 24.6 kb, with 21 exons, and the full-length cDNA is 4.8 kb, with an open reading frame for NtCaMK1 consisting of 1,415 amino acid residues. NtCaMK1 has all 11 subdomains of a kinase catalytic domain, lacks EF hands for Ca2+-binding, and is structurally similar to other CaMKs in mammal systems. Biochemical analyses have identified NtCaMK1 as a Ca2+/CaMK since NtCaMK1 phosphorylated itself and histone IIIs as substrate only in the presence of Ca2+/CaM with a Km of 44.5 microm and a Vmax of 416.2 nm min(-1) mg(-1). Kinetic analysis showed that the kinase not previously autophosphorylated had a Km for the synthetic peptide syntide-2 of 22.1 microm and a Vmax of 644.1 nm min(-1) mg(-1) when assayed in the presence of Ca2+/CaM. Once the autophosphorylation of NtCaMK1 was initiated, the phosphorylated form displayed Ca2+/CaM-independent behavior, as many other CaMKs do. Analysis of the CaM-binding domain (CaMBD) in NtCaMK1 with truncated and site-directed mutated forms defined a stretch of 20 amino acid residues at positions 913 to 932 as the CaMBD with high CaM affinity (Kd = 5 nm). This CaMBD was classified as a 1-8-14 motif. The activation of NtCaMK1 was differentially regulated by three tobacco CaM isoforms (NtCaM1, NtCaM3, and NtCaM13). While NtCaM1 and NtCaM13 activated NtCaMK1 effectively, NtCaM3 did not activate the kinase.     

Human calcium-calmodulin dependent protein kinase I: cDNA cloning, domain structure and activation by phosphorylation at threonine-177 by calcium-calmodulin dependent protein kinase I kinase.

Human Ca(2+)-calmodulin (CaM) dependent protein kinase I (CaMKI) encodes a 370 amino acid protein with a calculated M(r) of 41,337. The 1.5 kb CaMKI mRNA is expressed in many different human tissues and is the product of a single gene located on human chromosome 3. CaMKI 1-306, was unable to bind Ca(2+)-CaM and was completely inactive thereby defining an essential component of the CaM-binding domain to residues C-terminal to 306. CaMKI 1-294 did not bind CaM but was fully active in the absence of Ca(2+)-CaM, indicating that residues 295-306 are sufficient to maintain CaMKI in an auto-inhibited state. CaMKI was phosphorylated on Thr177 and its activity enhanced approximately 25-fold by CaMKI kinase in a Ca(2+)-CaM dependent manner. Replacement of Thr177 with Ala or Asp prevented both phosphorylation and activation by CaMKI kinase and the latter replacement also led to partial activation in the absence of CaMKI kinase. Whereas CaMKI 1-306 was unresponsive to CaMKI kinase, the 1-294 mutant was phosphorylated and activated by CaMKI kinase in both the presence and absence of Ca(2+)-CaM although at a faster rate in its presence. These results indicate that the auto-inhibitory domain in CaMKI gates, in a Ca(2+)-CaM dependent fashion, accessibility of both substrates to the substrate binding cleft and CaMKI kinase to Thr177. Additionally, CaMKI kinase responds directly to Ca(2+)-CaM with increased activity.     

Subcellular distributions of rat CaM kinase phosphatase N and other members of the CaM kinase regulatory system

Ca2+/Calmodulin-dependent protein kinase (CaM kinase) regulatory system is composed of multifunctional CaM kinases such as CaM kinases IV and I, upstream CaM kinases such as CaM kinase kinases alpha and beta, which activate multifunctional CaM kinases, and CaM kinase phosphatases such as CaM kinase phosphatase and CaM kinase phosphatase N, which deactivate the activated multifunctional CaM kinases. To understand the combinations of CaM kinases I and IV, CaM kinase kinases alpha and beta, and CaM kinase phosphatases, the locations of the enzymes in the cell were examined by immunocytochemical studies of cultured cells. The results indicate that CaM kinase I, CaM kinase kinase beta, and CaM kinase phosphatase occur in the cytoplasm and that CaM kinase IV, CaM kinase kinase alpha (and CaM kinase kinase beta in some cell types and tissues), and CaM kinase phosphatase N occur inside the cellular nucleus, suggesting that there are at least two different sets of CaM kinase regulatory systems, one consisting of CaM kinase I, CaM kinase kinase beta, and CaM kinase phosphatase in the cytoplasm and the other consisting of CaM kinase IV, CaM kinase kinase alpha (and CaM kinase kinase beta in some cell types and tissues), and CaM kinase phosphatase N in the nucleus.     

Functional analysis of Ca2+/calmodulin-dependent protein kinase II expressed in bacteria

The cDNA encoding the 50-kDa subunit of Ca2+/calmodulin (CaM)-dependent protein kinase II from adult rat brain was cloned into the bacterial expression vector pK223-2 and produced in bacteria. Extensive modification of the cDNA was required to express detectable levels of enzyme. The activity of the bacterially expressed kinase was stringently dependent on Ca2+/CaM but did not exhibit cooperative activation kinetics characteristic of the forebrain enzyme and required 10-fold greater amounts of CaM for half-maximal activation. The bacterially expressed enzyme displayed an apparent Km for a synthetic peptide substrate similar to that of the forebrain enzyme (12 and 10 microM, respectively). Limited proteolysis maps of autophosphorylated peptides, and Western blot analysis demonstrated that the bacterially expressed enzyme was structurally and immunologically indistinguishable from the 50-kDa subunit of the rat forebrain holoenzyme. The bacterially expressed enzyme became Ca2+/CaM-independent after Ca2+/CaM-dependent autophosphorylation in a fashion identical to the forebrain enzyme.     

Multiple Ca2+/calmodulin-dependent protein kinase genes in a unicellular eukaryote.

We purified a Ca2+/calmodulin (CaM)-dependent protein kinase (CaM kinase) from the yeast Saccharomyces cerevisiae with properties similar to mammalian type II CaM kinases. Degenerate oligonucleotides designed on the basis of the amino acid sequence of tryptic peptides from the 55 kd subunit of the yeast CaM kinase were used to isolate its gene from a set of lambda gt11-yeast genomic DNA phage clones initially selected by the ability to bind 125I-labelled yeast CaM. The cloned gene (CMK1) encodes an open reading frame that is homologous to the sequences of vertebrate type II CaM kinases. Several criteria demonstrated that the CMK1 gene product is the 55 kd polypeptide. Neither over-production (11-fold) nor complete elimination of the CMK1 gene product had any detectably deleterious effect on yeast cell growth. Extracts from cmk1 delta cells, which lacked detectable p55 using an antiserum raised against a Staphylococcus aureus protein A-CMK1 fusion protein, possessed significant residual Ca2+/CAM-dependent protein kinase activity. Using the CMK1 gene as a probe at low stringency, a second gene (CMK2) encoding another CaM-dependent protein kinase with striking sequence similarity to CMK1 was cloned. Deletion of CMK2, or both CMK1 and CMK2, was not lethal, although loss of CMK2 caused a slow rate of spore germination.     

A new approach for the detection of multiple protein kinases using monoclonal antibodies directed to the highly conserved region of protein kinases

To explore the protein kinase family enzymes expressed in cells, we attempted to generate antibodies that could detect a wide variety of protein kinases. For the production of such antibodies, synthetic peptides corresponding to amino acid sequences of a highly conserved subdomain (subdomain VIB) of the protein kinase family were used for immunization. Among the various peptide antigens, a peptide with 16 amino acids, CVVHRDLKPENLLLAS, effectively produced polyclonal antibodies with broad cross-reactivities to protein kinases. Two monoclonal antibodies, designated M8C and M1C, detected a variety of protein kinases such as calmodulin-dependent protein kinase II, calmodulin-dependent protein kinase IV, cAMP-dependent protein kinase, and mitogen-activated protein kinases, on Western blotting. The antibodies also immunoprecipitated various protein kinases in cell extracts. Furthermore, these antibodies could be used for detection of positive clones in the expression cloning of various protein kinases. Among 39 positive clones obtained from mouse brain cDNA library, 36 clones were identified as cDNA clones for various known and novel protein serine/threonine kinases, suggesting that the antibodies reacted highly specifically with various protein kinases. These results indicate that the present monoclonal antibodies directed to multiple protein kinases will be a powerful tool for the detection of a variety of known and novel protein kinases in cells.