Metastasis involves tumor cells moving through tissues and crossing tissue boundaries, which requires cell migration, remodeling of cell-to-cell contacts and interactions with the extracellular matrix. Individual tumor cells move in three-dimensional environments with either a rounded “ameboid” or an elongated “mesenchymal” morphology. These two modes of movement are tightly regulated by Rho family GTPases: elongated movement requires activation of Rac1, whereas rounded movement engages specific Cdc42 and Rho signaling pathways. It has been known for some time that events unfolding downstream of Ras GTPases are also involved in regulating multiple aspects of cell migration and invasion. More recently, RasGRF2—a Ras activator—has been identified as a suppressor of rounded movement, by inhibiting the activation of Cdc42, independently of its capacity to activate Ras. Here, we discuss how Rho and Ras signals can either cooperate or oppose each other in the regulation of cell migration and invasion.
Ras GTPases; Rho GTPases; RasGRF; cell migration and invasion; metastasis
When in the nucleus, ERK1/2 dislodges the retinoblastoma protein from lamin A, facilitating its rapid phosphorylation.
As orchestrators of essential cellular processes like proliferation, ERK1/2 mitogen-activated protein kinase signals impact on cell cycle regulation. A-type lamins are major constituents of the nuclear matrix that also control the cell cycle machinery by largely unknown mechanisms. In this paper, we disclose a functional liaison between ERK1/2 and lamin A whereby cell cycle progression is regulated. We demonstrate that lamin A serves as a mutually exclusive dock for ERK1/2 and the retinoblastoma (Rb) protein. Our results reveal that, immediately after their postactivation entrance in the nucleus, ERK1/2 dislodge Rb from its interaction with lamin A, thereby facilitating its rapid phosphorylation and consequently promoting E2F activation and cell cycle entry. Interestingly, these effects are independent of ERK1/2 kinase activity. We also show that cellular transformation and tumor cell proliferation are dependent on the balance between lamin A and nuclear ERK1/2 levels, which determines Rb accessibility for phosphorylation/inactivation.
Among the wealth of information that we have gathered about Ras in the past decade, the introduction of the concept of space in the field has constituted a major revolution that has enabled many pieces of the Ras puzzle to fall into place. In the early days, it was believed that Ras functioned exclusively at the plasma membrane. Today, we know that within the plasma membrane, the 3 Ras isoforms—H-Ras, K-Ras, and N-Ras—occupy different microdomains and that these isoforms are also present and active in endomembranes. We have also discovered that Ras proteins are not statically associated with these localizations; instead, they traffic dynamically between compartments. And we have learned that at these localizations, Ras is under site-specific regulatory mechanisms, distinctively engaging effector pathways and switching on diverse genetic programs to generate different biological responses. All of these processes are possible in great part due to the posttranslational modifications whereby Ras proteins bind to membranes and to regulatory events such as phosphorylation and ubiquitination that Ras is subject to. As such, space and these control mechanisms act in conjunction to endow Ras signals with an enormous signal variability that makes possible its multiple biological roles. These data have established the concept that the Ras signal, instead of being one single, homogeneous entity, results from the integration of multiple, site-specified subsignals, and Ras has become a paradigm of how space can differentially shape signaling.
Ras; GTPases; signal compartmentalization; acylation
RasGRF family guanine nucleotide exchange factors (GEFs) promote guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange on several Ras GTPases, including H-Ras and TC21. Although the mechanisms controlling RasGRF function as an H-Ras exchange factor are relatively well characterized, little is known about how TC21 activation is regulated. Here, we have studied the structural and spatial requirements involved in RasGRF 1/2 exchange activity on TC21. We show that RasGRF GEFs can activate TC21 in all of its sublocalizations except at the Golgi complex. We also demonstrate that TC21 susceptibility to activation by RasGRF GEFs depends on its posttranslational modifications: farnesylated TC21 can be activated by both RasGRF1 and RasGRF2, whereas geranylgeranylated TC21 is unresponsive to RasGRF2. Importantly, we show that RasGRF GEFs ability to catalyze exchange on farnesylated TC21 resides in its pleckstrin homology 1 domain, by a mechanism independent of localization and of its ability to associate to membranes. Finally, our data indicate that Cdc42-GDP can inhibit TC21 activation by RasGRF GEFs, demonstrating that Cdc42 negatively affects the functions of RasGRF GEFs irrespective of the GTPase being targeted.
Subcellular localization influences the nature of Ras/extracellular signal-regulated kinase (ERK) signals by unknown mechanisms. Herein, we demonstrate that the microenvironment from which Ras signals emanate determines which substrates will be preferentially phosphorylated by the activated ERK1/2. We show that the phosphorylation of epidermal growth factor receptor (EGFr) and cytosolic phospholipase A2 (cPLA2) is most prominent when ERK1/2 are activated from lipid rafts, whereas RSK1 is mainly activated by Ras signals from the disordered membrane. We present evidence indicating that the underlying mechanism of this substrate selectivity is governed by the participation of different scaffold proteins that distinctively couple ERK1/2, activated at defined microlocalizations, to specific substrates. As such, we show that for cPLA2 activation, ERK1/2 activated at lipid rafts interact with KSR1, whereas ERK1/2 activated at the endoplasmic reticulum utilize Sef-1. To phosphorylate the EGFr, ERK1/2 activated at lipid rafts require the participation of IQGAP1. Furthermore, we demonstrate that scaffold usage markedly influences the biological outcome of Ras site-specific signals. These results disclose an unprecedented spatial regulation of ERK1/2 substrate specificity, dictated by the microlocalization from which Ras signals originate and by the selection of specific scaffold proteins.
Sequestration of c-Fos at the nuclear envelope (NE) through interaction with A-type lamins suppresses AP-1–dependent transcription. We show here that c-Fos accumulation within the extraction-resistant nuclear fraction (ERNF) and its interaction with lamin A are reduced and enhanced by gain-of and loss-of ERK1/2 activity, respectively. Moreover, hindering ERK1/2-dependent phosphorylation of c-Fos attenuates its release from the ERNF induced by serum and promotes its interaction with lamin A. Accordingly, serum stimulation rapidly releases preexisting c-Fos from the NE via ERK1/2-dependent phosphorylation, leading to a fast activation of AP-1 before de novo c-Fos synthesis. Moreover, lamin A–null cells exhibit increased AP-1 activity and reduced levels of c-Fos phosphorylation. We also find that active ERK1/2 interacts with lamin A and colocalizes with c-Fos and A-type lamins at the NE. Thus, NE-bound ERK1/2 functions as a molecular switch for rapid mitogen-dependent AP-1 activation through phosphorylation-induced release of preexisting c-Fos from its inhibitory interaction with lamin A/C.
Ras proteins are distributed in distinct plasma-membrane microdomains and endomembranes. The biochemical signals generated by Ras therein differ qualitatively and quantitatively, but the extent to which this spatial variability impacts on the genetic program switched-on by Ras is unknown. We have used microarray technology to identify the transcriptional targets of localization-specific Ras subsignals in NIH3T3 cells expressing H-RasV12 selectively tethered to distinct cellular microenvironments. We report that the transcriptomes resulting from site-specific Ras activation show a significant overlap. However, distinct genetic signatures can also be found for each of the Ras subsignals. Our analyses unveil 121 genes uniquely regulated by Ras signals emanating from plasma-membrane microdomains. Interestingly, not a single gene is specifically controlled by lipid raft-anchored Ras. Furthermore, only 9 genes are exclusive for Ras signals from endomembranes. Also, we have identified 31 genes common to the site-specific Ras subsignals capable of inducing cellular transformation. Among these are the genes coding for Vitamin D receptor and for p120-GAP and we have assessed their impact in Ras-induced transformation. Overall, this report reveals the complexity and variability of the different genetic programs orchestrated by Ras from its main sublocalizations.
Ras; Compartmentalization; Gene microarrays; Transformation
Ras proteins are distributed in different types of plasma membrane microdomains and endomembranes. However, how microlocalization affects the signals generated by Ras and its subsequent biological outputs is largely unknown. We have approached this question by selectively targeting RasV12 to different cellular sublocalizations. We show here that compartmentalization dictates Ras utilization of effectors and the intensity of its signals. Activated Ras can evoke enhanced proliferation and transformation from most of its platforms, with the exception of the Golgi complex. Furthermore, signals that promote survival emanate primarily from the endoplasmic reticulum pool. In addition, we have investigated the need for the different pools of endogenous Ras in the conveyance of upstream mitogenic and transforming signals. Using targeted RasN17 inhibitory mutants and in physiological contexts such as H-Ras/N-Ras double knockout fibroblasts, we demonstrate that Ras functions at lipid rafts and at the Golgi complex are fully dispensable for proliferation and transformation.
Recent findings indicate that in addition to its location in the peripheral plasma membrane, H-Ras is found in endomembranes like the endoplasmic reticulum and the Golgi complex. In these locations H-Ras is functional and can efficiently engage downstream effectors, but little is known about how its activation is regulated in these environments. Here we show that the RasGRF family exchange factors, both endogenous and ectopically expressed, are present in the endoplasmic reticulum but not in the Golgi complex. With the aid of H-Ras constructs specifically tethered to the plasma membrane, endoplasmic reticulum, and Golgi complex, we demonstrate that RasGRF1 and RasGRF2 can activate plasma membrane and reticular, but not Golgi-associated, H-Ras. We also show that RasGRF DH domain is required for the activation of H-Ras in the endoplasmic reticulum but not in the plasma membrane. Furthermore, we demonstrate that RasGRF mediation favors the activation of reticular H-Ras by lysophosphatidic acid treatment whereas plasma membrane H-Ras is made more responsive to stimulation by ionomycin. Overall, our results provide the initial insights into the regulation of H-Ras activation in the endoplasmic reticulum.
Mxi2 is a p38α splice isoform that is distinctively activated by mitogenic stimuli. Here we show that Mxi2 immunoprecipitates carry a kinase activity that is persistently activated by epidermal growth factor in a fashion regulated by Ras, Raf, and MEK. We demonstrate that this kinase activity can be attributed not to Mxi2 but rather to extracellular signal-regulated kinases 1 and 2 (ERK1/2), which coimmunoprecipitated with Mxi2 both by ectopic expression and in a physiological environment like the kidney. Furthermore, we provide evidence that Mxi2-ERK interaction has profound effects on ERK function, demonstrating that Mxi2 prolongs the duration of the ERK signal by sustaining its phosphorylation levels. Interestingly, we show that the effects of Mxi2 on ERK are restricted to nuclear events. Mxi2 potently up-regulates ERK-mediated activation of the transcription factors Elk1 and HIF1α but has no effect on the activity of ERK cytoplasmic substrates RSK2 and cPLA2, induced by epidermal growth factor or by MEK. Overall, our findings point to Mxi2 as a unique member of the p38 family that may have an unprecedented role in the regulation of the functions of ERK mitogen-activated protein kinases.
The four receptor tyrosine kinases of the ErbB family play essential roles in several physiological processes and have also been implicated in tumor generation and/or progression. Activation of ErbB1/EGFR is mainly triggered by epidermal growth factor (EGF) and other related ligands, while activation of ErbB2, ErbB3, and ErbB4 receptors occurs by binding to another set of EGF-like ligands termed neuregulins (NRGs). Here we show that the Erk5 mitogen-activated protein kinase (MAPK) pathway participates in NRG signal transduction. In MCF7 cells, NRG activated Erk5 in a time- and dose-dependent fashion. The action of NRG on Erk5 was dependent on the kinase activity of ErbB receptors but was independent of Ras. Expression in MCF7 cells of a dominant negative form of Erk5 resulted in a significant decrease in NRG-induced proliferation of MCF7 cells. Analysis of Erk5 in several human tumor cell lines indicated that a constitutively active form of this kinase was present in the BT474 and SKBR3 cell lines, which also expressed activated forms of ErbB2, ErbB3, and ErbB4. Treatments aimed at decreasing the activity of these receptors caused Erk5 inactivation, indicating that the active form of Erk5 present in BT474 and SKBR3 cells was due to a persistent positive stimulus originating at the ErbB receptors. In BT474 cells expression of the dominant negative form of Erk5 resulted in reduced proliferation, indicating that in these cells Erk5 was also involved in the control of proliferation. Taken together, these results suggest that Erk5 may play a role in the regulation of cell proliferation by NRG receptors and indicate that constitutively active NRG receptors may induce proliferative responses in cancer cells through this MAPK pathway.