Joan Grindlay

The Bub2p spindle checkpoint links nuclear migration with mitotic exit.

Bfa1p and Bub2p are spindle checkpoint proteins that likely have GTPase activation activity and are associated with the budding yeast spindle pole body (SPB). Here, we show that Bfa1p and Bub2p bind the Ras-like GTPase Tem1p, a component of the mitotic exit network, to the cytoplasmic face of the SPB that enters the bud, whereas the GDP/GTP exchange factor Lte1p is associated with the cortex of the bud. Migration of the SPB into the bud probably allows activation of Tem1p through Lte1p, thereby linking nuclear migration with mitotic exit. Since components of the Bub2p checkpoint are conserved in other organisms, we propose that the position of the SPB or mammalian centrosome controls the timing of mitotic exit.

Rab25 and CLIC3 Collaborate to Promote Integrin Recycling from Late Endosomes/Lysosomes and Drive Cancer Progression

Summary Here we show that Rab25 permits the sorting of ligand-occupied, active-conformation α5β1 integrin to late endosomes/lysosomes. Photoactivation and biochemical approaches show that lysosomally targeted integrins are not degraded but are retrogradely transported and recycled to the plasma membrane at the back of invading cells. This requires CLIC3, a protein upregulated in Rab25-expressing cells and tumors, which colocalizes with active α5β1 in late endosomes/lysosomes. CLIC3 is necessary for release of the cell rear during migration on 3D matrices and is required for invasion and maintenance of active Src signaling in organotypic microenvironments. CLIC3 expression predicts lymph node metastasis and poor prognosis in operable cases of pancreatic ductal adenocarcinoma (PDAC). The identification of CLIC3 as a regulator of a recycling pathway and as an independent prognostic indicator in PDAC highlights the importance of active integrin trafficking as a potential drive to cancer progression in vivo.

The Mammalian MAPK/ERK Pathway Exhibits Properties of a Negative Feedback Amplifier

Analysis of ERK pathway circuitry suggests appropriate targets for inhibition, providing a guide for drug development. Biological Circuits Inform Drug Development The mitogen-activated protein kinase (MAPK) pathway involves a three-tiered kinase module, which amplifies the signal. Many cells also have negative feedback loops from the last kinase in the module to various points upstream in the pathway. Sturm et al. showed that, with negative feedback loops, the MAPK module results in a system like that of a negative feedback amplifier (NFA), which is an engineering design that smoothens the output to changes in input and makes a system robust to change. These NFA-like properties may explain why some cells are sensitive to inhibition of the second kinase in the cascade (they lack feedback loops), whereas other cells are resistant to inhibition at this point (their feedback loops are intact). These results also have implications for drug development, because inhibitors that target components that are outside the NFA are more effective at inhibiting the pathway. Three-tiered kinase modules, such as the Raf–MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase)–ERK (extracellular signal–regulated kinase) mitogen-activated protein kinase pathway, are widespread in biology, suggesting that this structure conveys evolutionarily advantageous properties. We show that the three-tiered kinase amplifier module combined with negative feedback recapitulates the design principles of a negative feedback amplifier (NFA), which is used in electronic circuits to confer robustness, output stabilization, and linearization of nonlinear signal amplification. We used mathematical modeling and experimental validation to demonstrate that the ERK pathway has properties of an NFA that (i) converts intrinsic switch-like activation kinetics into graded linear responses, (ii) conveys robustness to changes in rates of reactions within the NFA module, and (iii) stabilizes outputs in response to drug-induced perturbations of the amplifier. These properties determine biological behavior, including activation kinetics and the response to drugs.

Nud1p links astral microtubule organization and the control of exit from mitosis

The budding yeast spindle pole body (SPB) not only organizes the astral and nuclear microtubules but is also associated with a number of cell‐cycle regulators that control mitotic exit. Here, we describe that the core SPB component Nud1p is a key protein that functions in both processes. The astral microtubule organizing function of Nud1p is mediated by its interaction with the γ‐tubulin complex binding protein Spc72p. This function of Nud1p is distinct from its role in cell‐cycle control: Nud1p binds the spindle checkpoint control proteins Bfa1p and Bub2p to the SPB, and is part of the mitotic exit network (MEN) in which it functions upstream of CDC15 but downstream of LTE1. In conditional lethal nud1‐2 cells, the MEN component Tem1p, a GTPase, is mislocalized, whereas the kinase Cdc15p is still associated with the SPB. Thus, in nud1‐2 cells the failure of Tem1p to interact with Cdc15p at the SPB probably prevents mitotic exit.

Regulation of the Bfa1p–Bub2p complex at spindle pole bodies by the cell cycle phosphatase Cdc14p

The budding yeast mitotic exit network (MEN) is a GTPase-driven signal transduction cascade that controls the release of the phosphatase Cdc14p from the nucleolus in anaphase and thereby drives mitotic exit. We show that Cdc14p is partially released from the nucleolus in early anaphase independent of the action of the MEN components Cdc15p, Dbf2p, and Tem1p. Upon release, Cdc14p binds to the spindle pole body (SPB) via association with the Bfa1p–Bub2p GTPase activating protein complex, which is known to regulate the activity of the G protein Tem1p. Cdc14p also interacts with this GTPase. The association of the MEN component Mob1p with the SPB acts as a marker of MEN activation. The simultaneous binding of Cdc14p and Mob1p to the SPB in early anaphase suggests that Cdc14p initially activates the MEN. In a second, later step, which coincides with mitotic exit, Cdc14p reactivates the Bfa1p–Bub2p complex by dephosphorylating Bfa1p. This inactivates the MEN and displaces Mob1p from SPBs. These data indicate that Cdc14p activates the MEN in early anaphase but later inactivates it through Bfa1p dephosphorylation and so restricts MEN activity to a short period in anaphase.

Diacylglycerol kinase α controls RCP-dependent integrin trafficking to promote invasive migration

Phosphatidic acid generation by DGK-α is essential for the localization of Rab11-coupling protein to invasive pseudopods and subsequent invasive migration by tumor cells.

Phosphatase and feedback regulation of Raf-1 signaling.

The Raf-1 kinase is an effector of Ras GTPases that lies at the apex of the three-tier Raf/MEK/ERK pathway. Raf-1 activation is a complex process that entails two major events – relief of autoinhibition imposed by the regulatory domain and kinase domain activation. Recent studies indicate that the transition of Raf-1 from an active to an inactive state bears similar complexity to the activation process. Both these events require dynamic changes in Raf-1 phosphorylation. Here, we discuss the critical role of phosphatases and feedback phosphorylation during activation and inactivation of Raf-1 signalling.

A Raf-1 Mutant That Dissociates MEK/Extracellular Signal-Regulated Kinase Activation from Malignant Transformation and Differentiation but Not Proliferation

ABSTRACT It is widely thought that the biological outcomes of Raf-1 activation are solely attributable to the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. However, an increasing number of reports suggest that some Raf-1 functions are independent of this pathway. In this report we show that mutation of the amino-terminal 14-3-3 binding site of Raf-1 uncouples its ability to activate the MEK/ERK pathway from the induction of cell transformation and differentiation. In NIH 3T3 fibroblasts and COS-1 cells, mutation of serine 259 resulted in Raf-1 proteins which activated the MEK/ERK pathway as efficiently as v-Raf. However, in contrast to v-Raf, RafS259 mutants failed to transform. They induced morphological alterations and slightly accelerated proliferation in NIH 3T3 fibroblasts but were not tumorigenic in mice and behaved like wild-type Raf-1 in transformation assays measuring loss of contact inhibition or anchorage-independent growth. Curiously, the RafS259 mutants inhibited focus induction by an activated MEK allele, suggesting that they can hyperactivate negative-feedback pathways. In primary cultures of postmitotic chicken neuroretina cells, RafS259A was able to sustain proliferation to a level comparable to that sustained by the membrane-targeted transforming Raf-1 protein, RafCAAX. In contrast, RafS259A was only a poor inducer of neurite formation in PC12 cells in comparison to RafCAAX. Thus, RafS259 mutants genetically separate MEK/ERK activation from the ability of Raf-1 to induce transformation and differentiation. The results further suggest that RafS259 mutants inhibit signaling pathways required to promote these biological processes.

ERK2 drives tumour cell migration in three-dimensional microenvironments by suppressing expression of Rab17 and liprin-β2

Upregulation of the extracellular signal-regulated kinase (ERK) pathway has been shown to contribute to tumour invasion and progression. Because the two predominant ERK isoforms (ERK1 and ERK2, also known as MAPK3 and MAPK1, respectively) are highly homologous and have indistinguishable kinase activities in vitro, both enzymes were believed to be redundant and interchangeable. To challenge this view, we show that ERK2 silencing inhibits invasive migration of MDA-MB-231 cells, and re-expression of ERK2 but not ERK1 restores the normal invasive phenotype. A detailed quantitative analysis of cell movement on 3D matrices indicates that ERK2 knockdown impairs cellular motility by decreasing the migration velocity as well as increasing the time that cells spend not moving. Using gene expression arrays we found that the expression of the genes for Rab17 and liprin-β2 was increased by knockdown of ERK2 and restored to normal levels following re-expression of ERK2, but not ERK1. Both play inhibitory roles in the invasive behaviour of three independent cancer cell lines. Importantly, knockdown of either Rab17 or liprin-β2 restores invasiveness of ERK2-depleted cells, indicating that ERK2 drives invasion of MDA-MB-231 cells by suppressing expression of these genes.

Regulation of human myoblast differentiation by PEBP4

The RAF–MEK–ERK pathway regulates both myoblast proliferation and differentiation; however, it is unclear how these events are coordinated. Here, we show that human phosphatidylethanolamine‐binding protein 4 (PEBP4), a RAF kinase inhibitory protein (RKIP) family protein expressed preferentially in muscle, regulates the activity of the ERK pathway and myoblast differentiation by acting as a scaffold protein. In contrast to RKIP, which disrupts the RAF1–MEK interaction, PEBP4 forms ternary complexes with RAF1 and MEK, and can scaffold this interaction. PEBP4 expression is induced during the differentiation of primary human myoblasts. Consistent with the properties of a scaffold, PEBP4 enhances the RAF1–MEK interaction and the activation of MEK at low expression levels, whereas it inhibits these parameters at higher expression levels. Downregulation of PEBP4 by short hairpin RNA in human myoblasts increases MEK signalling and inhibits differentiation; by contrast, PEBP4 overexpression enhances differentiation. Thus, PEBP4 participates in the control of muscle cell differentiation by modulating the activity of MEK and ERK.