Atypical mitogen-activated protein kinases: structure, regulation and functions

Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that play a central role in transducing extracellular cues into a variety of intracellular responses ranging from lineage specification to cell division and adaptation. Fourteen MAP kinase genes have been identified in the human genome, which define 7 distinct MAP kinase signaling pathways. MAP kinases can be classified into conventional or atypical enzymes, based on their ability to get phosphorylated and activated by members of the MAP kinase kinase (MAPKK)/MEK family. Conventional MAP kinases comprise ERK1/ERK2, p38s, JNKs, and ERK5, which are all substrates of MAPKKs. Atypical MAP kinases include ERK3/ERK4, NLK and ERK7. Much less is known about the regulation, substrate specificity and physiological functions of atypical MAP kinases.     

Involvement of PPS3 phosphorylated by elicitor-responsive mitogen-activated protein kinases in the regulation of plant cell death

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant innate immunity. Overexpression of StMEK1(DD), a constitutively active MAPK kinase that activates salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), provokes hypersensitive response-like cell death in Nicotiana benthamiana. Here we purified a 51-kD MAPK, which was activated in potato (Solanum tuberosum) tubers treated with hyphal wall elicitor of a plant pathogen, and isolated the cDNA designated StMPK1. The deduced amino acid sequence of the StMPK1 showed strong similarity to stress-responsive MAPKs, such as tobacco (Nicotiana tabacum) SIPK and Arabidopsis (Arabidopsis thaliana) AtMPK6. To investigate the downstream signaling of StMPK1, we identified several proteins phosphorylated by StMPK1 (PPSs) using an in vitro expression cloning method. To dissect the biological function of PPSs in the plant defense, we employed virus-induced gene silencing (VIGS) in N. benthamiana. VIGS of NbPPS3 significantly delayed cell death induced by the transient expression of StMEK1(DD) and treatment with hyphal wall elicitor. Furthermore, the mobility shift of NbPPS3 on SDS-polyacrylamide gel was induced by transient expression of StMEK1(DD). The mobility shift of NbPPS3 induced by StMEK1(DD) was not compromised by VIGS of WIPK or SIPK alone, but drastically reduced by the silencing of both WIPK and SIPK. This work strongly supports the idea that PPS3 is a physiological substrate of StMPK1 and is involved in cell death activated by a MAPK cascade.     

Inactivation of mitogen-activated protein kinases by a mammalian tyrosine-specific phosphatase, PTPBR7

Mitogen-activated protein kinase (MAPK) is inactivated through dephosphorylation of tyrosyl and threonyl regulatory sites. In yeast, both dual-specificity and tyrosine-specific phosphatases are involved in dephosphorylation. In mammals, however, no tyrosine-specific phosphatase has been identified molecularly to dephosphorylate MAPK in vivo. Recently, we and others have cloned a murine tyrosine-specific phosphatase, PTPBR7/PTP-SL, which is expressed predominantly in the brain. Here we report inactivation of the extracellular signal-regulated kinase (ERK) family MAPK by PTPBR7. PTPBR7 made complexes with ERK1/ERK2 in vivo and dephosphorylated ERK1 in vitro. When overexpressed in mammalian cells, wild-type PTPBR7 suppressed the phosphorylation and activation of ERK by epidermal growth factor (EGF), nerve growth factor (NGF), and constitutively active MEK1, a mutant MAPK kinase. In contrast, catalytically inactive and ERK-binding-deficient mutants revealed little inhibition on the ERK cascade. These results indicate that PTPBR7 suppresses MAPK directly in vivo.     

Human T-cell mitogen-activated protein kinase kinases are related to yeast signal transduction kinases.

Mitogen-activated protein (MAP) kinase kinases, intermediates in a growth factor-stimulated protein kinase cascade, are dual specificity protein kinases that specifically phosphorylate and activate MAP kinases in response to extracellular signals. Here, we report the cloning of two forms of cDNA that encode this protein from human T-cells. MKK1a encodes a protein with predicted molecular size of 43,439 Da. Overexpression of this clone in COS cells led to elevated levels of protein and phorbol ester-stimulated MAP kinase kinase activity, confirming that MKK1a encodes the predicted protein. MKK1b, which appears to be an alternatively spliced form of the MKK1a gene, encodes a protein with predicted molecular size of 40,745 Da. Northern analysis revealed that the MKK1 cDNA hybridizes with a single 2.6-kilobase mRNA species in all human tissues examined. Sequence comparison shows homology to a group of yeast kinases that participate in signal transduction and to subdomain XI of other dual specificity kinase.     

Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

Background  

3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin) may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved.     

Yeast acetyl-CoA carboxylase: in vitro phosphorylation by mammalian and yeast protein kinases

Acetyl-CoA carboxylase (ACC) is regulated in mammalian tissues, in part, by multisite enzyme phosphorylation. Yeast ACC (Y-ACC) has been highly purified from S. cerevisiae by monomeric avidin-Sepharose chromatography, revealing an enzyme subunit species of molecular mass 265,000 Da. Unlike mammalian enzyme, Y-ACC is citrate-independent, and reacts weakly or not at all with a panel of anti-rat liver ACC antibodies. Like rat ACC, Y-ACC is rapidly phosphorylated and inactivated by two mammalian carboxylase kinases, the cAMP-dependent protein kinase and 5'-AMP-stimulated kinase. It is also phosphorylated by rat liver casein kinase II, but without any change in catalytic activity. Three major yeast protein kinases active on ACC have been fractionated; all co-elute with kinases active on casein, but each appears to be a distinct catalytic species. Like the mammalian casein kinases, however, phosphorylation of ACC by these yeast kinases does not alter yeast ACC activity. Taken together, these data indicate that Y-ACC possesses at least two classes of phosphorylation sites, one or more of which acutely regulates enzyme activity. Alterations in Y-ACC phosphorylation in yeast, as in mammalian tissues, could be an important modulator of the rates of fatty acid synthesis.     

Functional counterparts of mammalian protein kinases PDK1 and SGK in budding yeast

BACKGROUND: In animal cells, recruitment of phosphatidylinositol 3-kinase by growth factor receptors generates 3-phosphoinositides, which stimulate 3-phosphoinositide-dependent protein kinase-1 (PDK1). Activated PDK1 then phosphorylates and activates downstream protein kinases, including protein kinase B (PKB)/c-Akt, p70 S6 kinase, PKC isoforms, and serum- and glucocorticoid-inducible kinase (SGK), thereby eliciting physiological responses. RESULTS: We found that two previously uncharacterised genes of Saccharomyces cerevisiae, which we term PKH1 and PKH2, encode protein kinases with catalytic domains closely resembling those of human and Drosophila PDK1. Both Pkh1 and Pkh2 were essential for cell viability. Expression of human PDK1 in otherwise inviable pkh1Delta pkh2Delta cells permitted growth. In addition, the yeast YPK1 and YKR2 genes were found to encode protein kinases each with a catalytic domain closely resembling that of SGK; both Ypk1 and Ykr2 were also essential for viability. Otherwise inviable ypk1Delta ykr2Delta cells were fully rescued by expression of rat SGK, but not mouse PKB or rat p70 S6 kinase. Purified Pkh1 activated mammalian SGK and PKBalpha in vitro by phosphorylating the same residue as PDK1. Pkh1 activated purified Ypk1 by phosphorylating the equivalent residue (Thr504) and was required for maximal Ypk1 phosphorylation in vivo. Unlike PKB, activation of Ypk1 and SGK by Pkh1 did not require phosphatidylinositol 3,4,5-trisphosphate, consistent with the absence of pleckstrin homology domains in these proteins. The phosphorylation consensus sequence for Ypk1 was similar to that for PKBalpha and SGK. CONCLUSIONS: Pkh1 and Pkh2 function similarly to PDK1, and Ypk1 and Ykr2 to SGK. As in animal cells, these two groups of yeast kinases constitute two tiers of a signalling cascade required for yeast cell growth.     

Biphasic activation of two mitogen-activated protein kinases during the cell cycle in mammalian cells.

We studied mitogen-activated protein kinase (MAPK) activities during the cell cycle of Chinese hamster ovary (CHO) cells using site-specific antibodies against extracellular signal-regulated kinase-1, a 44-kDa MAPK (Boulton, T.G., Yancopoulos, G.D., Gregory, J.S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M.H. (1990) Science 249, 64-67). These antibodies detected two distinct MAPKs (44- and 42-kDa MAPKs) in CHO cells. CHO cells were arrested at metaphase in the M phase by treatment with nocodazole, and activities of MAPKs were analyzed at specific time points after release from arrest. Immune complex kinase assay and renaturation and phosphorylation assay in substrate-containing gel revealed that both 44- and 42-kDa MAPKs had activities in the G1 through S and G2/M phases and were activated biphasically, in the G1 phase and around the M phase. MAPKs were inactivated in metaphase-arrested cells. The amount of MAPKs did not change significantly in the cell cycle. In the G1, S, and G2/M phases, MAPKs were phosphorylated on both tyrosine and threonine residues and dephosphorylated in metaphase-arrested cells. Our data suggest that MAPKs may play some role in the cell cycle other than G0/G1 transition.     

Structurally unique yeast and mammalian serine-arginine protein kinases catalyze evolutionarily conserved phosphorylation reactions

The mammalian serine-arginine (SR) protein, ASF/SF2, contains multiple contiguous RS dipeptides at the C terminus, and approximately 12 of these serines are processively phosphorylated by the SR protein kinase 1 (SRPK1). We have recently shown that a docking motif in ASF/SF2 specifically interacts with a groove in SRPK1, and this interaction is necessary for processive phosphorylation. We previously showed that SRPK1 and its yeast ortholog Sky1p maintain their active conformations using diverse structural strategies. Here we tested if the mechanism of ASF/SF2 phosphorylation by SRPK is evolutionarily conserved. We show that Sky1p forms a stable complex with its heterologous mammalian substrate ASF/SF2 and processively phosphorylates the same sites as SRPK1. We further show that Sky1p utilizes the same docking groove to bind yeast SR-like protein Gbp2p and phosphorylates all three serines present in a contiguous RS dipeptide stretch. However, the mechanism of Gbp2p phosphorylation appears to be non-processive. Thus, there are physical attributes of SR and SR-like substrates that dictate the mechanism of phosphorylation, whereas the ability to processively phosphorylate substrates is inherent to SR protein kinases.     

Involvement of mitogen-activated protein kinases in adriamycin-induced cardiomyopathy

The current study investigated the phosphorylation of mitogen-activated protein kinases (MAPKs) as well as pro- and anti-apoptotic proteins in adriamycin (ADR)-induced cardiomyopathy (AIC) and heart failure in rats. Modulatory effects of antioxidant probucol on the activation of MAPKs were also examined. Male rats were administered with ADR (15 mg/kg body wt ip, over 2 wk) with and without probucol (120 mg/kg body wt for 4 wk ip). Hearts from these animals were studied at 1- to 24-h as well as at 3-wk posttreatment durations. In the 3-wk group, ADR depressed cardiac function, increased left ventricular end-diastolic pressure (LVEDP), and caused dyspnea and mortality. These changes were prevented by probucol. Phosphorylation of extracellular signal-regulated kinase (ERK)1/2, in the early stage of AIC, showed a biphasic response, with a maximum increase to 513% seen at 4 h, followed by a decrease to 66.8% at 3 wk after the last injection of ADR. Phosphorylation of p38 and c-Jun NH(2)-terminal kinases (JNKs) showed a steady increase through 2, 4, and 24 h and 3 wk (116% to 148%). In gene microarray analysis at 3 wk (heart failure stage), mRNA expression for both ERK1/2 and p38 kinases was decreased, whereas JNK mRNA was undetectable. Probucol completely prevented these MAPK changes. Activation of caspase-3 as well as the increase in the ratio of Bax to Bcl-xl were seen at early time points (1-24 h) as well as in the heart failure stage (3 wk). It is suggested that a transient increase in ERK1/2 at a shorter interval indicate an early adaptive response, and failure of this response corresponded with heart failure. In contrast, a gradual and persistent increase in p38 and JNK MAPKs as well as in caspase-3 and the Bax-to-Bcl-xl ratio may contribute in the initiation of apoptosis and progression of heart failure. Because probucol modulated changes in cellular signaling pathways and cardiac function, it is likely that oxidative stress plays a key role in AIC and heart failure.     

Mechanistic insights into Sky1p,a yeast homologue of the mammalian SR protein kinases

The SRPK family is distinguished from typical eukaryotic protein kinases by several unique structural features recently elucidated by X-ray diffraction methods [Nolen et al. (2001) Nat. Struct. Biol. 8, 176-183]. To determine whether these features impart unique catalytic function, the phosphorylation of the physiological Sky1p substrate, Npl3p, was monitored using steady-state and pre-steady-state kinetic techniques. While Sky1p has a low apparent affinity for ATP compared to other protein kinases, it binds Npl3p with very high affinity. The latter is achieved through a combination of local and distal factors in the protein substrate. The phosphoryl donor ATP has access to the nucleotide pocket in the absence or presence of Npl3p, indicating that a large protein substrate does not enforce an ordered addition of ligands. Sky1p binds two Mg(2+)-the first is essential whereas the second further enhances catalysis. While the turnover number is low (0.5 s(-1)), Npl3p is rapidly phosphorylated in the active site (40 s(-1)) based on single turnover experiments. These results indicate that Sky1p employs a catalytic pathway involving fast phosphoryl transfer followed by slow net release of products. These studies represent the first kinetic investigation of a member of the SRPK family and the first pre-steady-state kinetic study of a protein kinase using a natural protein substrate.     

Nutrient-regulated protein kinases in budding yeast

The ability of cells to react appropriately to nutritional cues is of fundamental importance, and in budding yeast, a small number of intracellular protein kinases, PKA, Snf1p/AMP-activated kinase, TOR, Gcn2p, and the cyclin-dependent kinase Pho85p have key roles. A recently characterized enzyme, PAS kinase, may be a new member of this group of nutritional transducers.     

Opposing regulation of B cell receptor-induced activation of mitogen-activated protein kinases by CD45

In this study, we examined the contribution made by CD45 to B cell antigen receptor (BCR)-induced activation of mitogen-activated protein kinase (MAPK) family members. We found that CD45 negatively regulated BCR-induced c-Jun NH(2)-terminal kinase (JNK) and p38 activation in immature WEHI-231 cells, whereas in mature BAL-17 cells, CD45 positively regulated JNK and p38 activation and negatively regulated extracellular signal-regulated kinase activity. Furthermore, cooperative action of JNK and p38 dictated BCR-induced inhibition of growth. Thus, CD45 appears to differentially regulate BCR-induced activation of MAPK members, and can exert opposing effects on JNK and p38 in different cellular milieu, controlling the B cell fate.