Sylvain Meloche

The mitogen-activated protein kinases (MAP kinases) p42mapk and p44mapk are serine/threonine kinases rapidly activated in cells stimulated with various extracellular signals by dual phosphorylation of tyrosine and threonine residues. They are thought to play a pivotal role in integrating and transmitting transmembrane signals required for growth and differentiation. Here we demonstrate that activation of these ubiquitously expressed MAP kinases is essential for growth. To specifically suppress MAP kinase activation in fibroblasts, we transiently expressed either the entire p44mapk antisense RNA or p44mapk kinase-deficient mutants (T192A or Y194F). As expected, and through independent mechanisms, both approaches strongly inhibited MAP kinase activation. The antisense reduced the expression of endogenous p42mapk and p44mapk by 90%, whereas overexpression of the T192A mutant inhibited growth factor activation of both endogenous MAP kinases by up to 70%. As a consequence, we found that the antisense as well as the T192A mutant of p44mapk inhibited growth factor-stimulated gene transcription (collagenase promoter assay with chloramphenicol acetyltransferase reporter) and cell growth. These effects were proportional to the extent of MAP kinase inhibition and reversed by coexpression of the wild-type p44mapk. Therefore we conclude that growth factor activation of p42mapk and p44mapk is an absolute requirement for triggering the proliferative response.

Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here, we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead, such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction, and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus, FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells, which fail to undergo either neural or mesodermal differentiation in adherent culture, and retain expression of pluripotency markers Oct4, Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for naïve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos.

Extracellular signal-regulated protein kinase (ERK, or mitogen-activated protein kinase [MAPK]) regulatory cascades in fungi turn on transcription factors that control developmental processes, stress responses, and cell wall integrity. CEK1 encodes a Candida albicans MAPK homolog (Cek1p), isolated by its ability to interfere with the Saccharomyces cerevisiae MAPK mating pathway. C. albicans cells with a deletion of the CEK1 gene are defective in shifting from a unicellular budding colonial growth mode to an agar-invasive hyphal growth mode when nutrients become limiting on solid medium with mannitol as a carbon source or on glucose when nitrogen is severely limited. The same phenotype is seen in C. albicans mutants in which the homologs (CST20, HST7, and CPH1) of the S. cerevisiae STE20, STE7, and STE12 genes are disrupted. In S. cerevisiae, the products of these genes function as part of a MAPK cascade required for mating and invasiveness of haploid cells and for pseudohyphal development of diploid cells. Epistasis studies revealed that the C. albicans CST20, HST7, CEK1, and CPH1 gene products lie in an equivalent, canonical, MAPK cascade. While Cek1p acts as part of the MAPK cascade involved in starvation-specific hyphal development, it may also play independent roles in C. albicans. In contrast to disruptions of the HST7 and CPH1 genes, disruption of the CEK1 gene adversely affects the growth of serum-induced mycelial colonies and attenuates virulence in a mouse model for systemic candidiasis.