Cellular adhesion via integrin receptors is intimately involved with regulation of signaling cascades. Phosphorylation of the Elk-1 transcription factor in response to growth factor treatment is impaired in nonadherent conditions. Under these conditions, growth factor activation of ERK is impaired. However, in these studies we bypassed this regulatory step by expression of active forms of Raf and MEK. Despite being able to render ERK activity anchorage independent, Elk-1 phosphorylation continued to display an adhesion requirement. Furthermore, we showed by confocal microscopy that when the ERK pathway is activated, nuclear translocation of ERK is hindered in suspended cells. Both localization of ERK from the cytoplasm into the nucleus and phosphorylation of Elk-1 were inhibited on CCD treatment, highlighting the importance of the integrin–actin cytoskeletal connection. By contrast, treatment with colchicine failed to abrogate 22W Raf–mediated phosphorylation of Elk-1, arguing against a requirement for an intact microtubule network in ERK translocation. Nuclear localization of ectopically expressed cyclin D1 was unaltered by disruption of the actin cytoskeleton, indicating that interference with nuclear localization is not a generalized effect. Thus, in addition to integrins being able to regulate growth factor activation of ERK, we suggest the presence of a second integrin-regulated checkpoint in the ERK cascade. This checkpoint is downstream of activation, but occurs at the level of active ERK accumulation in the nucleus and phosphorylation of its nuclear substrates.
The observation that integrity of the actin cytoskeleton is necessary for trafficking of ERK to the nucleus is a novel and interesting finding. Although it is well established that activation of the ERK pathway contributes to induction of cyclin D1, previous studies have yielded inconsistent results with regard to the role of ERK signaling in the adhesion-dependent expression of cyclin D1 expression. Roovers et al. 1999
reported that forced activation of the MEK/ERK pathway leads to the expression of cyclin D1 in suspended 3T3 cells, whereas Le Gall et al. 1998
, using a similar approach, failed to see cyclin D1 expression when the MEK/ERK pathway was activated in suspended CCL39 cells. As relatively low levels of ERK signaling are sufficient to induce cyclin D1 (for a review see Roovers and Assoian 2000
), our results may provide an explanation for these discrepant observations. Perhaps the different results obtained by Roovers et al. 1999
and Le Gall et al. 1998
reflects the fact that ERK translocation to the nucleus is strongly dependent on integrin signaling in some cell lines, whereas it is less strictly dependent on integrin signaling in others. Indeed, we do see low levels of nuclear ERK staining when induced tet-MEK*-3T3 cells used in Roovers et al. 1999
are cultured in suspension or treated with CCD.
We favor the explanation that integrins support efficient ERK translocation to the nucleus, as we find that in cells expressing active MEK, ERK preferentially colocalizes with MEK in the cytoplasm of nonadherent but not adherent cells. Other recent findings have pointed towards an adhesion dependence of ERK translocation to the nucleus (Danilkovitch et al. 2000
). In these studies, macrophage-stimulating protein showed reduced ERK activation and a further lack of detectable ERK translocation to the nucleus in suspended RE7 epithelial cells; however, this study did not examine ERK translocation under conditions where high ERK activity is maintained in suspended cells. ERK is deactivated by the activity of a variety of cellular phosphatases, including MKPs, and dephosphorylation of nuclear ERK leads to its rapid export (Khokhlatchev et al. 1998
), thus possibly presenting an alternative explanation of our results. However, in our system it is unlikely that dephosphorylation of ERK is upregulated in suspended cells, as ERK activation mediated by active versions of either Raf or MEK was unaltered in suspension versus adherent conditions. Additionally, under serum-free conditions levels of the nuclear-localized MKP-2 are unaltered in suspended versus adherent cells (data not shown). The activities controlling Elk-1 dephosphorylation are not well characterized, although recent studies in COS cells have implicated a role for the calcium-dependent protein phosphatase 2B (calcineurin) (Sugimoto et al. 1997
; Tian and Karin 1999
Integrin-mediated adhesion has been shown to recruit a variety of structural and signaling molecules into specialized sites and to cause the membrane localization of the GTPase, Rac (Burridge et al. 1992
; del Pozo et al. 2000
). However, the mechanism underlying effects of adhesion on ERK trafficking to the nucleus is as yet undetermined. ERK is sequestered in the cytoplasm through its interaction with binding partners, such as its upstream activator MEK, and efficient ERK-mediated activation of gene transcription is enhanced through the binding of the scaffolding protein, MEK partner 1 (MP-1) (Schaeffer et al. 1998
). Thus, the balance of ERK interactions between its upstream activators and scaffolding proteins may be altered by the state of cellular adhesion. Nuclear translocation of ERK is dependent on its ability to homodimerize; ERK mutants defective in this ability poorly translocate to the nucleus when microinjected into fibroblasts (Khokhlatchev et al. 1998
). An intriguing notion is that integrins, via the formation of an actin-based platform, enhance the ability of ERK monomers to homodimerize. Consistent with this idea, recent evidence suggests, at least under in vitro conditions, that ERK can directly bind to actin and actin-binding proteins, such as α-actinin (Leinweber et al. 1999
). Furthermore, active ERK molecules can be detected at sites of integrin-mediated adhesion (Fincham et al. 2000
). Future research will be directed at understanding the mechanism underlying the adhesion regulation of ERK nucleocytoplasmic trafficking.
Our findings add credence to the emerging theme that cell adhesion molecules regulate nuclear signaling events. Ingber and colleagues have shown that integrin “hard-wiring” is able to impact on nuclear structure (Maniotis et al. 1997
). Further, certain integrins may provide direct modulation of nuclear events. For example, engagement of the leukocyte integrin LFA-1/αLβ2 has been shown to initially bind and subsequently promote the nuclear localization of the c-Jun coactivator, JAB1, leading to enhanced activating protein 1 (AP-1) transcriptional activity (Bianchi et al. 2000
). High expression levels of the cell–cell adhesion molecules, E- and N-cadherin, reduce the nuclear localization and transcription potential of β-catenin, by recruiting it to sites of cell–cell contact (Sadot et al. 1998
; Orsulic et al. 1999
). In conjunction with our current findings on integrin regulation of ERK localization, these other reports highlight an important role for cell adhesion molecules and the actin cytoskeleton in the nuclear trafficking of signaling molecules.