Growth factor activation of MAP kinase requires cell adhesion

Abstract

The MAP kinase pathway is a major regulator of both normal and oncogenic growth. We report that activation of the MAP kinase ERK2 by serum or purified growth factors is strongly dependent on cell adhesion to extracellular matrix proteins. This effect is specific to soluble growth factors, since suspended cells still activate ERK2 in response to plating on fibronectin, and is reversible. Analysis of endogenous Ras and Raf show that these proteins are still activated by serum in suspended cells, whereas MEK activity is inhibited. Conversely, activation of ERK2 by activated mutants of Ras and Raf is still adhesion‐dependent but activation by MEK is not. Consistent with these results, activated MEK enhances growth of ras‐transformed cells in suspension but not when adherent. These results identify a novel synergism between cell adhesion‐ and growth factor‐regulated pathways, and explain how oncogenic activation of MAP kinases induces both serum‐ and anchorage‐independent growth.

Integrins in regulation of tissue development and function

“…In doing so, integrins support the cyclin Ecdk2 activity that drives S-phase entry [31,32]. The enhanced and sustained Erk activity in adherent cells (see above) explains in part the supportive role of integrins in cyclin D1 transcription [16,33]. Many pathways that connect integrins to Erk activation have been implicated in cell proliferation, such as the assembly of protein complexes including Src, FAK, and p130Cas [10] or including Fyn, caveolin, and Shc [34].…”

“…For instance, growth factors and integrinmediated cell adhesion can each independently trigger a weak transient activation of the Erk-type mitogenactivated protein kinase but the combined effect of cell adhesion and growth factors is a strong and sustained Erk activity. Signalling by integrins and growth factor receptors in this situation converges at the level of Raf or MEK [15,16].…”

The c-Abl tyrosine kinase contributes to the transient activation of MAP kinase in cells plated on fibronectin

“…Integrins contribute to the sustained activation of Erks by growth factors (Renshaw et al, 1997;Lin et al, 1997;Miyamoto et al, 1996), which is important for cell cycle progression. However, the activation of Erks when cells are replated on FN is highly transient and is therefore unlikely to promote progression through G1.…”

“…At 24 h after transfection, cells were switched to DMEM with 0.5% calf serum for an additional 24 h. When indicated, cells were detached and held in suspension for 2 h then replated on dishes coated with FN as described above. Cells were lysed in bu er with 1% NP40, 0.25 M NaCl and inhibitors (Renshaw et al, 1997); endogenous Erk2 was immunoprecipitated using polyclonal anti-Erk2 (Santa Cruz Biotechnology, Santa Cruz CA, USA) or transfected HA-tagged Erk2 immunoprecipitation with 12CA5. One third of the precipitates was analysed for Erk2 protein by Western blotting with anti-Erk2 antibody.…”

“…One third of the precipitates was analysed for Erk2 protein by Western blotting with anti-Erk2 antibody. The remaining two-thirds was assayed for Erk2 activity using an in-gel kinase assay as described (Renshaw et al, 1997;Kamashita and Fujisawa 1989). Bands were quanti®ed by scanning densitometry and Erk activity normalized for variations in protein to determine the speci®c activity against chicken Src.…”