Cross-talk of calcium-dependent protein kinase and MAP kinase signaling

Abstract

Plants use different signaling pathways to acclimate to changing environmental conditions. Fast changes in the concentration of free Ca²⁺ ions—so called Ca²⁺ signals—are among the first responses to many stress situations. These signals are decoded by different types of calcium-dependent protein kinases, which—together with mitogen-activated protein kinases (MAPK)—present two major pathways that are widely used to adapt the cellular metabolism to a changing environment. Ca²⁺-dependent protein kinase (CDPK) and MAPK pathways are known to be involved in signaling of abiotic and biotic stress in animal, yeast and plant cells. In many cases both pathways are activated in response to the same stimuli leading to the question of a potential cross-talk between those pathways. Cross-talk between Ca²⁺-dependent and MAPK signaling pathways has been elaborately studied in animal cells, but it has hardly been investigated in plants. Early studies of CDPKs involved in the biotic stress response in tobacco indicated a cross-talk of CDPK and MAPK activities, whereas a recent study in Arabidopsis revealed that CDPKs and MAPKs act differentially in innate immune signaling and showed no direct cross-talk between CDPK and MAPK activities. Similar results were also reported for CDPK and MAPK activities in the salt-stress response in Arabidopsis. Different modes of action are furthermore supported by the different subcellular localization of the involved kinases. In this review, we discuss recent findings on CDPK and MAPK signaling with respect to potential cross-talk and the subcellular localization of the involved components.

Plants are constantly exposed to changing environmental conditions to which they must adapt in order to survive. Many extracellular stimuli like abiotic or biotic stress lead to fast and transient changes in the intracellular free Ca²⁺ concentration.¹ These Ca²⁺ fluctuations are decoded by different calcium-binding proteins and protein kinases.^2–5 In addition, other protein kinases such as mitogen-activated protein kinases (MAPKs) or Snf1-related kinases (SnRKs) become activated in response to the same stimuli as well.^6–8 This raises the question to what extent these different signaling pathways influence each other, what we define as cross-talk here.

Decoding of Ca²⁺ signals is mediated by different gene families in plants: By Ca²⁺-dependent protein kinases (CDPKs) and CDPK-related kinases (CRKs), by calmodulines (CAMs) and calmodulin-dependent protein kinases (CaMKs), by calcineurin B-like proteins (CBLs) and CBL interacting protein kinases (CIPKs, also called SnRK3s), and by Ca²⁺- and calmodulin-dependent protein kinases (CCaMKs). CCaMKs are present as single gene in a number of plant species engaging symbiotic interactions but not in Arabidopsis.^2,3 CDPKs, CRKs and CCaMKs combine Ca²⁺-sensing and decoding within one molecule by fusion of a protein kinase domain with a C-terminal Ca²⁺-binding domain. CDPKs are present only in plants, ciliates and apicomplexan parasites including the malaria parasite, Plasmodium falciparum.⁹ As this unique family of protein kinases regulates a number of essential pathways in those parasites they present bona fide drug targets and have therefore been intensively studied. Accordingly, the first complete CDPK structure was recently solved from apicomplexan parasites.¹⁰ The canonical CDPK comprises a calcium-binding domain of four EF-hands attached to the C-terminal end of a Ser/Thr protein kinase domain with an interjacent autoinhibitory junction domain. The binding of Ca²⁺ to the C-terminal EF-hands induces a large conformational change resulting in relief of inhibition of the kinase activity.¹⁰ MAPK pathways present protein kinase cascades composed of a MAP kinase kinase kinase (MKKK), a MAP kinase kinase (MKK) and a MAPK.¹¹ MAPKs act as last component in a protein kinase cascade, and one of their major tasks is to transduce an extracellular stimulus into a transcriptional response in the nucleus.

Subcellular Localization—Where the Kinase Hits Its Targets

The kinase domain and the C-terminal Ca²⁺-binding domain of plant CDPKs are strongly conserved, only the N-terminal domain is highly variable and contains information for their subcellular targeting for example by N-myristoylation and palmitoylation, which is predicted for 27 out of the 34 CDPKs in Arabidopsis.⁴ N-terminal acylation of CDPKs has been reported to be required for their attachment to specific cellular membranes.^12–15 The same mechanism of subcellular targeting also applies to other Ca²⁺ decoding systems like the CBL-CIPK system, where it has also been demonstrated that the membrane attachment is essential for the biological function of the signaling pathway, for example in the salt-stress response.^16,17

In contrast, only four out of the 20 MAPKs in Arabidopsis have a glycine in position two in their amino acid sequence, which is an absolute requirement for N-myristoylation,¹⁸ and only AtMPK20 is predicted to be N-myristoylated by different prediction program like the myristoylator (expasy.org/tools/myristoylator) or the NMT-MYR predictor (mendel.imp.ac.at/myristate/).¹⁹

Accordingly, the MAPKs studied so far were mainly reported to be localized in the cytosol and the nucleus (Table 1) where they interact with components of the transcriptional machinery and also with regulatory phosphatases.^20–22 Plant MAPKs were also found to be associated with microtubules as regulators of cell morphology and polarization in response to stresses as well as during plant growth and development.²³ However, despite their lack of obvious molecular mechanisms for membrane attachment, some MAPKs have also been detected in membrane fractions in different proteomic approaches.⁸ Moreover, AtMPK6 was recently found to be localized at the plasma membrane and the trans-Golgi network.²⁴ In different stress-response pathways the major task of MAPK pathways is to translate an extra-cellular stimulus into an appropriate cellular response, either by transcriptional induction of stress responsive genes for long-term adaptation or by direct regulation of enzymatic activities or channel proteins in the immediate stress response.²⁵ This pathway of signal transduction from the plasma membrane to the nucleus or cytosolic targets is reflected by the localization of the involved kinases. Whereas many MKKKs have been identified in different proteomics studies of membranes (reviewed in ref. 8), only few data on membrane-localized MAPKs have been published. The rather complex localization pattern of MAPKs does also reflect their multiple roles in cellular stress response and regulation of growth and development and most importantly enables them to interact with a great number of different targets. In line with their N-terminal acylation, most of the CDPKs have been identified at different cellular membranes, either as GFP-fusion proteins^13,14 and also in proteomics approaches (Table 1).^26,27

Table 1.

Localization and functions of selected Arabidopsis CDPKs and MAPKs

Protein	Gene identifier	Subcellular localization	Function	Ref
CDPKs:
AtCPK 1	At5g04870	peroxisomes, oilbodies	cold stress, pathogen defence	13, 63, 64
AtCPK 3	At4g23650	cytosol, nucleus, membrane	salt stress, drought stress, pathogen response	13, 15
AtCPK 4	At4g09570	cytosol, nucleus	drought stress, pathogen response	13, 22
AtCPK 5	At4g35310	cytosol, nucleus	drought stress, pathogen response	22
AtCPK 6	At2g17290	plasma membrane, nucleus, cytosol	drought stress, pathogen response	14, 22
AtCPK 11	At1g35670	cytosol, nucleus	pathogen response	22
AtCPK 13	At3g51850	nucleus, cytosol, plasma membrane	pathogen response	14
AtCPK 21	At4g04720	membrane	drought stress?	13
AtCPK 23	At4g04740	plasma membrane	drought stress	60
AtCPK 32	At3g57530	nucleus, cytosol	ABA signaling	28
MAPKs:
AtMPK1	At1g10210		pathogen response	40
AtMPK3	At3g45640	nucleus (parsely)	pathogen response	21
AtMPK4	At4g01370	nucleus	salt stress, cold stress	20
AtMPK6	At2g43790	nucleus, cytosol, plasma membrane TGN	salt stress, cold stress, pathogen response	20, 24
AtMPK7	At2g18170		pathogen response	40
AtMPK9	At3g18040	nucleus, and cytosol	ROS-mediated ABA signaling	56
AtMPK12	At2g46070	nucleus, and cytosol	ROS-mediated ABA signaling	56

However, also complex localization patterns, which are similar to MAPKs, have been reported for some CDPKs. For example, AtCPK32, a regulator of ABA-responsive gene expression, is localized in the nucleus and at the plasma membrane²⁸ and AtCPK3, which is involved in salt stress acclimation, is localized at the plasma and the vacuolar membrane, and also in the nucleus and the cytosol.^13,15 Consistently, potential CPK3 targets are localized in the cytosol like nitrate reductase or a phospholipase A^29,30 and at the plasma membrane and the vacuole.¹⁵ In the cytosol both CDPKs and MAPKs have been shown to regulate metabolic enzymes by direct phosphorylation. In the case of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), the rate-limiting enzyme of ethylene biosynthesis, the phosphorylation by MAPKs and by CDPKs affects protein stability and turnover.^31,32

Cross-talk between CDPK and MAPK Pathways in Animals

Cross-talk between Ca²⁺ and MAPK signaling is well known in animal cells, where Ca²⁺ signals and calmodulines (CaMs) regulate the Ras/Raf/ERK-MAP kinase pathway.^33,34 Notably, quite different forms of cross-talk between Ca²⁺ and MAPK signaling pathways have been observed. In the common view, Ca²⁺ signals activate the MAPKs ERK and p38 in response to external signals,³³ and a direct activation of the MEK/ERK MAP kinase pathway by CaMKII in the regulation of cell cycle progression has been reported in reference ³⁵. However, also cases in which Ca²⁺ and CaM have a clear inhibitory effect on ERK activation have been described in reference ³³ and ³⁴, as well as a “reversed” regulation of a non-canonical Wnt/cyclic GMP/Ca²⁺ pathway by an upstream p38 MAPK pathway.³⁶ Considering this complex picture of regulation and cross-talk of the ERK MAPK pathway with Ca²⁺-signaling in animal cells it becomes clear that the common view that MAPK cascades are downstream of Ca²⁺ signaling pathways cannot be generalized. It rather seems that cross-talk between Ca²⁺-signaling and MAPK pathways has to be specifically considered for each individual stimulus and the involved kinases.³⁷

CDPK-MAPK Cross-talk and Cross-tolerance between Biotic- and Abiotic-Stress Responses in Plants

CDPKs and MAP kinase pathways have been identified as central components mediating plant immunity.^38–42 In early studies addressing cross-talk between CDPK and MAPK signaling in response to biotic stress it was found that the expression of a deregulated version of tobacco NtCDPK2 results in activation of defence reactions and cell death in infiltrated leaves.⁴³ This deregulated version of NtCDPK2 was lacking its C-terminal regulatory autoinhibitory and Ca²⁺-binding domains and was therefore constitutively active as protein kinase. The responses included the synthesis of reactive oxygen species, induction of defence genes and cell death. Furthermore, the expression of the deregulated NtCDPK2 triggered the production of the phytohormones jasmonic acid and ethylene, but not salicylic acid and compromised stress-induced MAPK activation in tobacco. On the other hand, a recent comprehensive study of Arabidopsis CDPKs demonstrated that the expression of deregulated CDPKs did not affect the activities of AtMPK3 and 6, the two major players in the plant immune response.²² Moreover, gene expression analysis revealed that CDPKs and MAPK cascades act differentially in the immune response. Analysis of the expression levels of several flg22 inducible genes in mesophyll protoplasts expressing deregulated AtCPK5 and AtMKK4 identified MAPK specific, CDPK specific, CDPK/MAPK synergistic and MAPK dominant target genes. Thus, also in plants different levels of cross-talk between Ca²⁺-dependent and MAPK pathways exist.

From accumulating data it becomes clear that a cellular function of one particular kinase can not be attributed exclusively to a single type of stress response. Several CDPKs and MAPKs have been found to be involved in the biotic as well as in the abiotic stress response. For example, AtMKK2 has been identified as a key regulator of the cold- and salt-stress response in Arabidopsis⁴⁴ but was also found to be involved in the response to Pseudomonas syringae infections.⁴⁵ AtCPK3, which recently turned out to be involved in salt-stress acclimation,¹⁵ has also been identified together with AtCPK13 as part of the herbivore response signaling.⁴⁶ AtCPK3 and AtCPK13 knock out mutants accumulated equal levels of the phytohormones jasmonic acid (JA), abscisic acid (ABA) and ethylene as the wild type in response to infection, indicating that AtCPK3 and AtCPK13 are not upstream regulators of phytohormone biosynthesis and thus seem to act differently to NtCDPK2. Moreover, AtCPK3 was also able to induce the flagellin-responsive NHL10 reporter in transient leaf protoplast assays.²² In contrast, AtCPK3 had no effect on the transcriptional induction of known salt stress-responsive genes and on the activation of AtMPK4 and 6 in the salt-stress response, also indicating different modes of action for AtCPK3 and AtMPK4 and 6 in the salt-stress response.¹⁵

Often the response to one type of stress renders plants more resistant to another type of stress, a phenomenon called cross-tolerance.⁴⁷ Both, MAPKs and CDPKs have been implicated in cross-tolerance between biotic and abiotic stress responses. Wounding or overexpression of pathogen-induced MYB transcription factors increases salt tolerance in tomato⁴⁸ and CDPK activities were found to be involved in this cross-tolerance.⁴⁹ In Arabidopsis, the expression of active AtMKK9 induces ethylene and camalexin biosynthesis and increases salt sensitivity.⁵⁰

Initially, many MAPKs and CDPKs have been cloned based on their identification as stress-responsive genes or in functional genetic screens for altered stress tolerance.⁵¹ In addition, the high conservation of these signaling pathways between yeast and plants could be used for isolation and first functional analysis of stress-responsive genes.^52–54 The plant hormone ABA is well-known for its important role in mediating abiotic stress responses⁵¹ and both CDPKs and MAPKs have been implicated in ABA-responsive gene expression.^28,55,56 Recently, it has been found that ABA also plays an important role in the biotic stress response where it acts as a negative regulator of disease resistance⁵⁷ but can also promote plant defense in some plant-pathogen interactions.⁵⁸ ABA is also involved in the aforementioned cross-tolerance between biotic and abiotic stress responses.⁴⁸ The function of ABA to control stomatal closure is another example for a complex regulatory network of CDPK and MAPK pathways. Initially, AtCPK3 and 6 were linked to ABA- and Ca²⁺-induced stomatal closure, and recently AtCPK21 and 23 were identified as regulators of the guard cell anion channel SLAC1.^59,60 AtMPK9 and 12, which are preferentially and highly expressed in guard cells, also function as positive regulators of stomatal closure, and ABA and Ca²⁺ signals cannot activate anion channels in mpk9/12 mutants, thus indicating that these two MAPKs act between the ABA and Ca²⁺ signals and the anion channels.⁵⁶ However, whether the activities of AtMPK9/12 are influenced by AtCPK21/23 or by AtCPK3/6, or vice versa, remains to be tested.

Conclusions and Perspectives

Considering pathways involving both MAPKs and CDPKs, it becomes clear that signaling pathways have to be envisaged rather as complex networks than as linear or branched signaling pathways. Moreover, some elements function in multiple response pathways, which could be more or less independent from each other, or work in a cooperative manner. Analysis of the subcellular localization of the involved components could help to classify the type of response. The immediate early response covers the initiation of signaling cascades or pathways towards altered gene expression in the nucleus and starts at the plasma membrane. This immediate response also includes the regulation of membrane proteins such as ion channels and cytosolic proteins. Accordingly membrane-bound kinases like CDPKs are ideally suited for this purpose. In the following steps the signals have to be transduced through the cytosol into the nucleus to start the long-term adaptation by induction of gene expression. This task requires soluble kinases and could involve MAPKs (and MKKs) as well as some CDPKs. The fact that stress-responsive genes have been identified as being MAPK specific, CDPK specific, CDPK/MAPK synergistic or MAPK dominant target genes shows that these different pathways could act in parallel. In addition, it has to be considered that protein expression and turnover is also regulated beyond the transcriptional level. Also here both principal signaling pathways—MAPKs and CDPKs—are involved. For example in response to salt- or osmotic stress the regulation of general protein synthesis by MAPKs and a CAMK-like protein kinase has been described in yeast and plants.^61,62

Abbreviations

ABA

abscisic acid

CDPK

calcium-dependent protein kinase

MAPK

mitogen-activated protein kinase

CAM

calmoduline

JA

jasmonic acid

Snf1

sucrose non-fermenting 1