The activation of MAPKs is controlled by the balance between MAPK kinase and MAPK phosphatase activities. The latter is mediated by a subset of phosphatases with dual specificity (VH-1 family). Here, we describe a new member of this family encoded by the puckered gene of Drosophila. Mutations in this gene lead to cytoskeletal defects that result in a failure in dorsal closure related to those associated with mutations in basket, the Drosophila JNK homolog. We show that puckered mutations result in the hyperactivation of DJNK, and that overexpression of puc mimics basket mutant phenotypes. We also show that puckered expression is itself a consequence of the activity of the JNK pathway and that during dorsal closure, JNK signaling has a dual role: to activate an effector, encoded by decapentaplegic, and an element of negative feedback regulation encoded by puckered. In many cases, cell differentiation represents a transition between two states of cellular activity-one in which cells proliferate and acquire information about their fates and identities, and another in which cells stop dividing and manifest the information gathered earlier.Many of the signaling pathways leading to cell differentiation depend on phosphorylation cascades. Mounting evidence points to signaling through MAP kinase (MAPK) pathways as a key component in this transition. Three distinct types of MAPK pathways have been identified: p42-p44 ERKs (extracellular signal-related kinases), p38 kinases, and p46-p54 JNKs (Jun N (amino)-terminal kinases). These major subfamilies transduce signals from different stimuli. The ERKs respond predominantly to growth factors and hormones and are activated in a Ras-dependent manner. The p38 and JNKs respond to different environmental stresses and are activated preferentially downstream of Rac1 and Cdc42 small G proteins (for review, see Canman and Kastan 1996). In most cases, MAPK activation is a transient event, even in the continuing presence of the stimulus that leads to its activation. MAPK activity is controlled by the balance of MAPK kinase and MAPK phosphatase activities.The dorsal closure of the Drosophila embryo provides an example of cell differentiation and how this is usually coupled to morphogenetic events and movements that shape late stages in development. Half way through embryogenesis, the dorsal surface of the embryo is covered by an extraembryonic membrane, the amnioserosa, which contacts the epidermis. After proliferation stops, the epidermis stretches dorsally and, as it encroaches the amnioserosa, closes the existing gap. Three phases lead to the successful completion of this event. The dorsalward movement of the epidermal cells, an anteroposterior stretching of the embryo and the seaming of the dorsal epidermis (Martinez-Arias 1993). The completion of this process takes several hours and is associated with specialized behavior of the dorsal-most epidermal cells. These cells display planar polarity reflected in the arrangement of the cytoskeleton, which is essential for the normal process...

Endothelial cells respond to vascular endothelial growth factor (VEGF) to produce new blood vessels. This process of angiogenesis makes a critical contribution during embryogenesis and also in the response to ischemia in adult tissues. We have studied the intracellular trafficking of the major VEGF receptor KDR (VEGFR2). Unlike other related growth factor receptors, we find that a significant proportion of KDR is held in an endosomal storage pool within endothelial cells. We find that KDR can be delivered to the plasma membrane from this intracellular pool and that VEGF stimulates this recycling to the cell surface. KDR recycling appears to be distinct from the previously characterized Rab4-and Rab11-dependent pathways, but, instead, KDR ؉ recycling vesicles contain Src tyrosine kinase and VEGFstimulated recycling requires Src activa- IntroductionAngiogenesis is the fundamental physiologic process by which new blood vessels are generated from preexisting vasculature. It plays a crucial role in embryogenesis, where it is required for elaboration of the vasculature from the primary vascular plexus. In normal adult physiology, angiogenesis is significant in a relatively limited number of processes-primarily in the formation of endometrial vessels in the uterus and development of the corpus luteum during the ovulation cycle. 1 Angiogenesis becomes more widely important to adult physiology through its critical involvement in a number of pathologic conditions. Active angiogenesis makes a positive contribution to the woundhealing process and also in the response to tissue ischemia-hypoxic tissues generate proangiogenic signals that stimulate the formation of new vessels and so improve perfusion. 2 Angiogenesis makes an unwanted contribution to the growth of solid tumors, with cancer cells secreting proangiogenic factors to provide a blood supply for the growing mass. 3 Deregulated angiogenesis is an underlying cause of proliferative diabetic retinopathy, a major vascular complication of both type I and type II diabetes, 4 and also contributes to other disease states such as age-related macular degeneration, rheumatoid arthritis, and psoriasis. 5,6 Understanding of the regulatory mechanisms of angiogenesis is hence seen not only as a fundamental problem in human biology but also as an important goal in medical research.Angiogenesis is controlled by a wide range of positive and negative signals. Vascular endothelial growth factor (VEGF) is the most critical and potent of the proangiogenic regulators 7-9 and is secreted by tissues in response to hypoxia or inflammation. 9 VEGF binds to VEGF receptors (VEGFRs) on the surface of endothelial cells, triggering a cascading series of signaling pathways that stimulate endothelial cell sprouting, migration, tube formation, proliferation, and survival. 10 There are 3 human members of the VEGFR family: VEGFR1/Flt-1, VEGFR2/Flk-1/KDR, and VEGFR3/Flt-4. 11 These proteins are members of the larger family of receptor tyrosine kinases (RTKs), which includes the plateletderived growth f...

Members of the Rho family of small GTPases control cell adhesion and motility through dynamic regulation of the actin cytoskeleton. Although twelve family members have been identified, only three of these - RhoA, Rac and Cdc42 - have been studied in detail. RhoA regulates the formation of focal adhesions and the bundling of actin filaments into stress fibres. It is also involved in other cell signalling pathways including the regulation of gene expression and the generation of lipid second messengers [1] [2]. RhoA is very closely related to two other small GTPases about which much less is known: RhoB and RhoC (which are approximately 83% identical). Perhaps the most intriguing of these is RhoB. RhoA is largely cytosolic but translocates to the plasma membrane on activation. RhoB, however, is entirely localised to the cytosolic face of endocytic vesicles [3] [4]. This suggests a potential role for RhoB in regulating endocytic traffic; however, no evidence has been presented to support this. RhoA has been shown to act at the plasma membrane to regulate the clathrin-mediated internalisation of transferrin receptor [5] and of the muscarinic acetylcholine receptor [6]. We have recently demonstrated that RhoB binds the RhoA effector, PRK1 and targets it to the endosomal compartment [7]. We show here that RhoB acts through PRK1 to regulate the kinetics of epidermal growth factor receptor traffic.

A fundamental control point in the regulation of the initiation of protein synthesis is the formation of the eukaryotic initiation factor 4F (eIF-4F) complex. The formation of this complex depends upon the availability of the mRNA cap binding protein, eIF-4E, which is sequestered away from the translational machinery by the tight association of eIF-4E binding proteins (4E-BPs). Phosphorylation of 4E-BP1 is critical in causing its dissociation from eIF-4E, leaving 4E available to form translationally active eIF-4F complexes, switching on mRNA translation. In this report, we provide the first evidence that the phosphorylation of 4E-BP1 increases during mitosis and identify Ser-65 and Thr-70 as phosphorylated sites. Phosphorylation of Thr-70 has been implicated in the regulation of 4E-BP1 function, but the kinase phosphorylating this site was unknown. We show that the cyclin-dependent kinase, cdc2, phosphorylates 4E-BP1 at Thr-70 and that phosphorylation of this site is permissive for Ser-65 phosphorylation. Crucially, the increased phosphorylation of 4E-BP1 during mitosis results in its complete dissociation from eIF-4E.

The Rho family of small GTPases play a pivotal role in the dynamic regulation of the actin cytoskeleton. Recent studies have suggested that these signalling proteins also have wide-ranging functions in membrane trafficking pathways. The Rho family member RhoB was shown to localise to vesicles of the endocytic compartment, suggesting a potential function in regulation of endocytic traffic. In keeping with this, we have previously shown that expression of active RhoB causes a delay in the intracellular trafficking of the epidermal growth factor (EGF) receptor; however, the site of action of RhoB within the endocytic pathway is still unknown. RhoB exists as two prenylated forms in cells: geranylgeranylated RhoB (RhoB-GG) and farnesylated RhoB (RhoB-F). Here we use farnesyltransferase inhibitors (FTIs) to show that prenylation specifies the cellular localisation of RhoB. RhoB-GG localises to multivesicular late endosomes and farnesylated RhoB (RhoB-F) localises to the plasma membrane. The gain of endosomal RhoB-GG elicited by FTI treatment reduces sorting of EGF receptor to the lysosome and increases recycling to the plasma membrane. Ultrastructural analysis shows that activation of RhoB through drug treatment or mutation has no effect the sorting of receptor into late endosomes, but instead inhibits the subsequent transfer of late endosomal receptor to the lysosome.