Angel R. Nebreda

A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins

MAPK-activated protein kinase-2 (MAPKAP kinase-2) is activated in vitro by the p42 and p44 isoforms of MAPK (p42/p44MAPK). In several cell lines, however, MAPKAP kinase-2 is activated by sodium arsenite, heat shock, or osmotic stress and not by agonists that activate p42/p44MAPK. We have identified a MAPK-like enzyme that acts as a MAPKAP kinase-2 reactivating kinase (RK). RK is recognized by an antiserum raised against a Xenopus MAPK (Mpk2), which is most similar to HOG1 from S. cerevisiae. We also identified a RK kinase (RKK) on the basis of its ability to activate either RK or a GST-Mpk2 fusion protein. The RKK, RK, and MAPKAP kinase-2 constitute a new stress-activated signal transduction pathway in vertebrates that is distinct from the classical MAPK cascade.

Mechanisms and functions of p38 MAPK signalling

The p38 MAPK (mitogen-activated protein kinase) signalling pathway allows cells to interpret a wide range of external signals and respond appropriately by generating a plethora of different biological effects. The diversity and specificity in cellular outcomes is achieved with an apparently simple linear architecture of the pathway, consisting of a core of three protein kinases acting sequentially. In the present review, we dissect the molecular mechanisms underlying p38 MAPK functions, with special emphasis on the activation and regulation of the core kinases, the interplay with other signalling pathways and the nature of p38 MAPK substrates as a source of functional diversity. Finally, we discuss how genetic mouse models are facilitating the identification of physiological functions for p38 MAPKs, which may impinge on their eventual use as therapeutic targets.

p38 MAP kinases: beyond the stress response.

We are grateful to Anne Ephrussi and Giulio Superti-Furga for comments on the manuscript. We apologize to the authors whose original work is not included in the references owing to space limitations.

Essential Role of p38α MAP Kinase in Placental but Not Embryonic Cardiovascular Development

p38alpha MAP kinase is activated in response to many cellular stresses and also regulates the differentiation and/or survival of various cell types in vitro, including skeletal muscle cells and cardiomyocytes. Here we show that targeted inactivation of the mouse p38alpha gene results in embryonic lethality at midgestation correlating with a massive reduction of the myocardium and malformation of blood vessels in the head region. However, this defect appears to be secondary to insufficient oxygen and nutrient transfer across the placenta. When the placental defect was rescued, p38alpha(-/-) embryos developed to term and were normal in appearance. Our results indicate that p38alpha is required for placental organogenesis but is not essential for other aspects of mammalian embryonic development.

A link between MAP kinase and p34cdc2/cyclin B during oocyte maturation: p90rsk phosphorylates and inactivates the p34cdc2 inhibitory kinase Myt1

M‐phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34cdc2. In G2‐arrested Xenopus oocytes, there is a stock of p34cdc2/cyclin B complexes (pre‐MPF) which is maintained in an inactive state by p34cdc2 phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34cdc2 inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2→M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M‐phase entry in Xenopus oocytes, but its precise contribution to the activation of pre‐MPF is unknown. Here we show that the C‐terminal regulatory domain of Myt1 specifically binds to p90rsk, a protein kinase that can be phosphorylated and activated by MAPK. p90rsk in turn phosphorylates the C‐terminus of Myt1 and down‐regulates its inhibitory activity on p34cdc2/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK–p90rsk cascade can trigger some Myt1 phosphorylation prior to pre‐MPF activation. We found that Myt1 preferentially associates with hyperphosphorylated p90rsk, and complexes can be detected in immunoprecipitates from mature oocytes. Our results suggest that during oocyte maturation MAPK activates p90rsk and that p90rsk in turn down‐regulates Myt1, leading to the activation of p34cdc2/cyclin B.

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs

During studies of the activation and inactivation of the cyclin B‐p34cdc2 protein kinase (MPF) in cell‐free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose‐binding protein and the Xenopus c‐mos protein kinase (malE‐mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE‐mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE‐mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c‐mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified.

p38α MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation

Stem cell function is central for the maintenance of normal tissue homeostasis. Here we show that deletion of p38α mitogen-activated protein (MAP) kinase in adult mice results in increased proliferation and defective differentiation of lung stem and progenitor cells both in vivo and in vitro. We found that p38α positively regulates factors such as CCAAT/enhancer-binding protein that are required for lung cell differentiation. In addition, p38α controls self-renewal of the lung stem and progenitor cell population by inhibiting proliferation-inducing signals, most notably epidermal growth factor receptor. As a consequence, the inactivation of p38α leads to an immature and hyperproliferative lung epithelium that is highly sensitized to K-RasG12V-induced tumorigenesis. Our results indicate that by coordinating proliferation and differentiation signals in lung stem and progenitor cells, p38α has a key role in the regulation of lung cell renewal and tumorigenesis.

Feedback control of the protein kinase TAK1 by SAPK2a/p38α

TAB1, a subunit of the kinase TAK1, was phosphorylated by SAPK2a/p38α at Ser423, Thr431 and Ser438 in vitro. TAB1 became phosphorylated at all three sites when cells were exposed to cellular stresses, or stimulated with tumour necrosis factor‐α (TNF‐α), interleukin‐1 (IL‐1) or lipopolysaccharide (LPS). The phosphorylation of Ser423 and Thr431 was prevented if cells were pre‐incubated with SB 203580, while the phosphorylation of Ser438 was partially inhibited by PD 184352. Ser423 is the first residue phosphorylated by SAPK2a/p38α that is not followed by proline. The activation of TAK1 was enhanced by SB 203580 in LPS‐stimulated macrophages, and by proinflammatory cytokines or osmotic shock in epithelial KB cells or embryonic fibroblasts. The activation of TAK1 by TNF‐α, IL‐1 or osmotic shock was also enhanced in embryonic fibroblasts from SAPK2a/p38α‐deficient mice, while incubation of these cells with SB 203580 had no effect. Our results suggest that TAB1 participates in a SAPK2a/p38α‐mediated feedback control of TAK1, which not only limits the activation of SAPK2a/p38α but synchronizes its activity with other signalling pathways that lie downstream of TAK1 (JNK and IKK).

Regulation of the meiotic cell cycle in oocytes.

The mitotic and meiotic cell cycle share many regulators, but there are also important differences between the two processes. The meiotic maturation of Xenopus oocytes has proved useful for understanding the regulation of Cdc2-cyclin-B, a key activator of G2/M progression. New insights have been made recently into the signalling mechanisms that induce G2-arrested oocytes to resume and complete the meiotic cell cycle.

Xkid, a chromokinesin required for chromosome alignment on the metaphase plate.

Metaphase chromosome alignment is a key step of animal cell mitosis. The molecular mechanism leading to this equatorial positioning is still not fully understood. Forces exerted at kinetochores and on chromosome arms drive chromosome movements that culminate in their alignment on the metaphase plate. In this paper, we show that Xkid, a kinesin-like protein localized on chromosome arms, plays an essential role in metaphase chromosome alignment and in its maintenance. We propose that Xkid is responsible for the polar ejection forces acting on chromosome arms. Our results show that these forces are essential to ensure that kinetochores and chromosome arms align on a narrow equatorial plate during metaphase, a prerequisite for proper chromosome segregation.