Four p38 MAPK isoforms have been characterized, namely p38α, p38β, p38γ, and p38δ, which have in common a 12-amino-acid activation loop containing a TGY motif located at amino acid position 180 to 182. CD4 T cells predominantly express the p38α and p38δ isoforms . Activation of p38 MAPK occurs by the phosphorylation of Thr180 and Tyr182, leading to conformational reorganization of the enzyme and binding of ATP and the phosphoryl acceptor (substrate). Two different sequential binding mechanisms for ATP and the substrate have been proposed [15,16], although the order in which ATP and the phosphoryl acceptor bind may occur randomly and may depend on the phosphoryl acceptor .
The rate-limiting step in the kinetic mechanism of p38 MAPK activation is still unknown but may be of great importance in designing new inhibitors of p38. More than 100 different p38 MAPK inhibitors have been reported so far, and all are competitive with ATP. However, and in contrast to ATP, these compounds can bind to both the active and inactive (unphosphorylated) forms of p38, providing an advantage over ATP and resulting in a very potent inhibitory capacity, regardless of high intracellular ATP concentrations . Since the first generation of p38 MAPK inhibitors, like the pyridinyl imidazole compound SB203580, which have been shown to affect several unrelated kinases, the understanding of the kinome has greatly improved and has facilitated the development of more selective inhibitors . These new molecules have now helped to clarify the role of p38 MAPK in vitro and to define the mechanisms by which p38 MAPK controls for example LPS-induced cytokine expression in macrophages .
Activation of p38 MAPK
The MAPK pathway is similar for all the members of the MAPK family and is typically composed of a highly conserved MAPK module comprising three kinases, namely MAPK kinase kinase (MKKK), MAPK kinase (MKK), and MAPK  (Fig. 1). In the p38 MAPK cascade, MEKK4 (an MKKK) activates MKK3, MKK4, or MKK6, which subsequently phosphorylate p38 MAPK at Thr180 and Tyr182 [21,22] (Fig. 1). The mechanisms regulating the activation of the MAPK module are very complex, in particular in T cells in which the TCR and CD28 act synergistically to induce intracellular cell signaling.
One critical event after stimulation of the TCR that is essential for activation of the MAPK signal cascade is the recruitment of linker for activation of T cells (LAT) and the activation of guanine nucleotide exchange factors (GEFs). GEFs activate small GTP-binding proteins such as Ras, Rac-1, and Cdc42 by promoting the conversion of the GDP-bound inactive state to the GTP-bound active state, leading to the activation of the MAPK signaling cascade. Activation of the p38 MAPK cascade in Jurkat T cells has been shown to require phosphorylation of the GEF Vav by Zap-70 and subsequent activation of Rac-1. CD28 co-stimulation augments the recruitment of Vav to LAT and Zap-70 and increases Zap-70 mediated Vav phosphorylation . Rac-1 elicits the p38 MAPK cascade through the p21-activated kinase 1 (Pak1), although the exact mechanism remains unclear because Pak1 does not directly activate an MKKK .
Direct upstream activators of MKKKs are the growth arrest and DNA damage-inducible genes 45 (GADD45) proteins, which are important in the regulation of p38 MAPK activity in T cells [25,26]. GADD45 proteins can bind the autoinhibitory domain of MEKK4 (MKKK), which is an upstream activator of p38 MAPK and JNK, and relieve the autoinhibition of MEKK4, leading to activation of the MAPK cascade . Whether GADD45 proteins are activated by Pak1 remains to be elucidated. Interestingly, the activation of p38 MAPK by cytokines seems to occur in two phases that can be regulated by two different mechanisms: a rapid but brief GADD45β-independent activation followed by a delayed but sustained GADD45β-dependent activation [28,29]. Although data on the role and mode of activation of GADD45 proteins in T cells are still controversial, the regulation of the expression levels of GADD45 proteins constitutes an indirect additional mechanism to control the intensity and duration of p38 MAPK activation.
An alternative pathway for p38 MAPK activation in T cells has been recently described in which dual phosphorylation of Thr180 and Tyr182 is not induced by an MKK but by p38 MAPK itself. Stimulation of the TCR induces phosphorylation of p38 MAPK on Tyr323 through Zap70, which subsequently leads to autophosphorylation of Thr180 and Tyr182 . It has been suggested that in T cells, the classical pathway in which GADD45 proteins activate MEKK4 might be induced predominantly by stress signals, whereas the alternative pathway might be activated by TCR stimulation . However, p38 MAPK phosphorylation induced by TCR ligation is impaired in GADD45β-deficient naive T cells , indicating that both the classical pathway and the alternative pathway are required for p38 MAPK activation in T cells (Fig. 1).
Inactivation of p38 MAPK
The level of protein phosphorylation is controlled by the coordinated activities of kinases and phosphatases. Dephosphorylation of either Thr180 and Tyr182 is sufficient to inactivate p38 MAPK and can be mediated by tyrosine-specific MAPK phosphatases (TS-MKPs) such as phospho-tyrosine phosphatase SL (PTP-SL), serine/threonine-specific MKPs (SS-MKPs) such as protein phosphatase type 2A (PP2A), or tyrosine and threonine dual-specificity phosphatases (DS-MKPs) such as MKP1. The MAPK cascade can induce phosphatase gene transcription, providing a negative feedback for MAPK activation . Another mechanism of inactivation of p38 MAPK is mediated by GADD45α, which has recently been shown to inhibit the activation of p38 MAPK by the alternative pathway, but not by the classical pathway, in T cells but not in B cells  (Fig. 1). Similarly, GADD45β can inhibit the JNK pathway by binding one of its upstream activators, MKK7 . These observations are intriguing because GADD45 proteins are, as mentioned above, also activators of the MAPK signaling cascade and therefore seem to be important in regulating p38 MAPK activity by exerting both activating and inactivating effects.
Substrates of p38 MAPK
All the MAPKs phosphorylate a threonine or a tyrosine, which is immediately followed by a proline residue. This 'P + 1' sequence is the most reliable consensus motif for MAPK substrates . The specificity of the different members of the MAPK family and of the different isoforms of p38 MAPK is provided by a docking motif usually composed of three domains: the basic region, the LXL motif, and the hydrophobic region. The hydrophobic region seems to be of particular importance for the determination of the substrate specificity for p38 MAPK . The development of models to predict p38 MAPK docking-domain specificities may permit the design of inhibitory peptides to block the phosphorylation of specific subsets of substrates so as to block specific pathways mediated by p38 MAPK .
p38 MAPK substrates can be divided into two categories, namely transcription factors and protein kinases (Table 1). Several of the protein kinases activated by p38 MAPK are involved in the control of gene expression at different levels. Mitogen- and stress-activated kinase 1 and 2 (MSK1/2), for example, can directly activate transcription factors such as cAMP-response element-binding protein (CREB), activating transcription factor 1 (ATF1), NF-κB p65, signal transducers and activators of transcription (STAT1), and STAT3 [38-41], but can also phosphorylate the nucleosomal proteins histone H3 and high-mobility-group 14 (HMG-14). Either by inducing chromatin remodeling or by recruiting the transcriptional machinery, these two proteins are important for the rapid induction of immediate-early genes that occurs in response to stress or mitogenic stimuli . In contrast to MSK1/2, which preferentially activates transcription, MAP kinase-activated protein kinase 2 (MK2) participates in the control of gene expression at the post-transcriptional level by phosphorylating tristetraprolin (TTP) or heat shock protein 27 (hsp27) .
Stimuli activating p38 MAPK
Environmental stress such as osmotic shock activates p38 MAPK in almost every mammalian cell. A variety of other stimuli, such as cell-cell contact or soluble factors such as cytokines, are also able to activate p38. In T cells, the p38 MAPK is activated by contact with APCs or by different cytokines. Triggering of the TCR alone leads to activation of the p38 MAPK pathway in naive and memory CD4 T cells. However, several reports have demonstrated that full activation of p38 MAPK in vitro requires co-stimulation in addition to TCR stimulation. The co-stimulatory molecules CD28, 4-1BB, CD26, CD30, inducible co-stimulator (ICOS), and erythropoietin-producing hepatocyte B6 (EphB6) have been shown to activate p38 MAPK synergistically with TCR stimulation [44-50]. Interestingly, ligation of CD30, CD28, or EphB6 also activates p38 MAPK in the absence of TCR ligation [46,47,49,51]. However, the requirement for p38 MAPK activation with regard to co-stimulatory receptor ligation differs between T cell subsets. Whereas the p38 MAPK pathway can be activated by CD28 stimulation alone in memory CD4 T cells, naive T cells strictly require concomitant TCR signaling , indicating that naive T cells are lacking an important molecule necessary to link the CD28 signaling to the p38 MAPK signaling cascade. This deficiency might contribute to the higher activation threshold of naive T cells than that of memory T cells.
In addition to co-stimulatory molecules, some cytokine receptors can activate p38 MAPK in T cells. The IL-12 receptor, for example, has been shown to signal by means of the p38 MAPK cascade in activated T cells. However, activation of p38 MAPK by IL-12 alone is only transient (less than 20 minutes) . Sustained activation of p38 MAPK can be observed by simultaneous stimulation with IL-12 and IL-18 and requires the expression of GADD45β . Whether IL-12/IL-18 directly activates GADD45β or simply induces its expression remains a matter of debate [26,28]. IL-4 and IL-2 have been shown to induce p38 MAPK activation in the murine T cell line CT6 but not in primary T cells [51,53,54]. In our hands, IL-4 was unable to activate p38 MAPK in primary naive and memory human CD4 T cells (F Dodeller, A Skapenko, H Schulze-Koops, unpublished data). This could be related to the relatively low expression of the IL-4 receptor in primary cells in comparison with the murine cell line, but also to differences in the signaling pathway of IL-4 in primary human T cells and T cell lines . Interestingly, p38 MAPK has been implicated in IL-4 receptor signaling in human airway smooth muscle cells  and in murine B cells .