Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regu-lates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration.

Objective-Vascular endothelial growth factor receptor 2 (VEGFR2) is a receptor tyrosine kinase that regulates vascular physiology. However, mechanism(s) by which VEGFR2 signaling and trafficking is coordinated are not clear. Here, we have tested endocytic Rab GTPases for regulation of VEGFR2 trafficking and signaling linked to endothelial cell migration. Methods and Results-Quiescent VEGFR2 displays endosomal localization and colocalization with the Rab5a GTPase, an early endosome fusion regulator. Expression of GTP or GDP-bound Rab5a mutants block activated VEGFR2 trafficking and degradation. Manipulation of Rab7a GTPase activity associated with late endosomes using overexpression of wild-type or mutant proteins blocks activated VEGFR2 trafficking and degradation. Depletion of Rab7a decreased VEGFR2 Y1175 phosphorylation but increased p42/44 (pERK1/2) MAPK phosphorylation. Endothelial cell migration was increased by Rab5a depletion but decreased by Rab7a depletion. Conclusions-Rab5a and Rab7a regulate VEGFR2 trafficking toward early and late endosomes. Our data suggest that VEGFR2-mediated regulation of endothelial function is dependent on different but specific Rab-mediated GTP hydrolysis activity required for endosomal trafficking. Key Words: VEGFR2 Ⅲ Rab Ⅲ trafficking Ⅲ signaling Ⅲ migration V ascular endothelial growth factor A (VEGF-A) is an essential regulator of vascular physiology. 1 VEGF-A plays key roles in vasculogenesis and angiogenesis during development. 2 Angiogenesis is needed for embryogenesis and wound healing but is also associated with cancer, eye diseases, and other chronic disorders. The importance of VEGF-A is underlined by the fact that deletion of a single mouse VEGF-A allele causes embryonic lethality attributable to the impairment of vascular development. 3,4 The VEGF-A gene gives rise to at least 6 to 12 different variants, with VEGF-A 165 (termed VEGF-A) being the most abundant and proangiogenic isoform. 5,6 VEGF-A can bind to 2 different but closely related endothelial receptor tyrosine kinases (RTKs): fms-like tyrosine kinase (VEGFR1 or Flt-1) and kinase insert domain receptor (VEGFR2 or KDR). A major proangiogenic axis is based on VEGF-A binding to VEGFR2, stimulating tyrosine kinase activity, autophosphorylation, and signaling to downstream target proteins which regulate diverse processes including chemotaxis, migration, vascular permeability, cell survival, and proliferation. 7 Such physiological responses are regulated via multiple effectors including focal adhesion kinase (FAK), c-Akt/PKB, extracellular-signal related kinase (ERK) pathways, and stimulation of nitric oxide and prostaglandin synthesis. 8 Whereas VEGFR2 signaling pathways that regulate endothelial responses are well characterized, the spatial and temporal order underlying such events remains elusive. For example, VEGFR2 plasma membrane activation leads to trafficking to endosomes and degradation in lysosomes. 9 Simultaneously, VEGFR2 activation leads to tyrosine phosphorylation and intracellular signaling. 7,8 H...

Various members of the canonical family of transient receptor potential channels (TRPCs) exhibit increased cation influx following receptor stimulation or Ca 2؉ store depletion. Tyrosine phosphorylation of TRP family members also results in increased channel activity; however, the link between the two events is unclear. We report that two tyrosine residues in the C terminus of human TRPC4 (hTRPC4), Tyr-959 and Tyr-972, are phosphorylated following epidermal growth factor (EGF) receptor stimulation of COS-7 cells. This phosphorylation was mediated by Src family tyrosine kinases (STKs), with Fyn appearing to be the dominant kinase. In addition, EGF receptor stimulation induced the exocytotic insertion of hTRPC4 into the plasma membrane dependent on the activity of STKs and was accompanied by a phosphorylation-dependent increase in the association of hTRPC4 with Na ؉ /H ؉ exchanger regulatory factor. Furthermore, this translocation and association was defective upon mutation of Tyr-959 and Tyr-972 to phenylalanine. Significantly, inhibition of STKs was concomitant with a reduction in Ca 2؉ influx in both native COS-7 cells and hTRPC4-expressing HEK293 cells, with cells expressing the Y959F/Y972F mutant exhibiting a reduced EGF response. These findings represent the first demonstration of a mechanism for phosphorylation to modulate TRPC channel function.The precise regulation of Ca 2ϩ signaling is crucial to the function and survival of all cell types, with disrupted Ca 2ϩ handling contributing to the pathogenesis of many diseases, including heart failure, ischemia, and neuronal degeneration (1-3). Control is maintained through complex interplays between plasma membrane proteins, the mitochondria, endoplasmic reticulum, Ca 2ϩ -binding proteins, and second messenger systems (4, 5). A ubiquitous mechanism of Ca 2ϩ entry is through the activation of non-voltage-gated Ca 2ϩ channels located in the plasma membrane. These channels respond to either receptor activation or intracellular Ca 2ϩ store depletion to control events such as gene expression, secretion, and migration (6). Various members of the transient receptor potential (TRP) 2 family of cation channels are implicated as important components of this ubiquitous Ca 2ϩ signaling pathway and represent an important area for potential drug therapy (7).The canonical or classic transient receptor potential ion channel subfamily (TRPC) is one of three TRP subfamilies known to mediate receptor-operated and store-operated Ca 2ϩ entry (7). The most compelling experimental evidence for a role of a TRPC protein in store-operated Ca 2ϩ entry came from studies on the TRPC4 null mouse where TRPC4 was identified as an essential component of store-operated Ca 2ϩ channels in aortic endothelial cells (8). Evidence for a role of TRPC channels in receptor-operated Ca 2ϩ entry include the finding that isolated endothelial cells from the TRPC4 knock-out mouse exhibited reduced Ca 2ϩ entry in response to acetylcholine, ATP, and thrombin application, and antisense oligonucleotide down-re...

Angiotensin II (AII, 100 nM) stimulation of bovine adrenal chromaf®n cells (BACCs) produced angiotensin II receptor subtype 1 (AT 1 )-mediated increases in extracellular regulated protein kinase 1/2 (ERK1/2) and stress-activated p38MAPK (p38 kinase) phosphorylation over a period of 10 min. ERK1/2 and p38 kinase phosphorylation preceded Ser31 phosphorylation on tyrosine hydroxylase (TOH). The inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) activation, PD98059 (0.1±50 mM) and UO126 (0.1±10 mM), dosedependently inhibited both ERK2 and Ser31 phosphorylation on TOH in response to AII, suggesting MEK1/2 involvement. The p38 kinase inhibitor SB203580 (20 mM, 30 min) abolished Ser31 and Ser19 phosphorylation on TOH and partially inhibited ERK2 phosphorylation produced by AII. In contrast, 1 mM SB203580 did not affect AII-stimulated TOH phosphorylation, but fully inhibited heat shock protein 27 (HSP27) phosphorylation produced by AII. Also, 1 mM SB203580 fully inhibited Ser19 phosphorylation on TOH and HSP27 phosphorylation in response to anisomycin (30 min, 10 mg/mL). The results suggest that ERKs mediate Ser31 phosphorylation on TOH in response to AII, but p38 kinase is not involved. Previous studies suggesting a role for p38 kinase in the phosphorylation of Ser31 are explained by the non-speci®c effects of 20 mM SB203580 in BACCs. The p38 kinase pathway is able to phosphorylate Ser19 on TOH in response to anisomycin, but does not do so in response to AII.

Rab GTPases are implicated in endosome-to-plasma membrane recycling, but how such membrane traffic regulators control vascular endothelial growth factor receptor 2 (VEGFR2/KDR) dynamics and function are not well understood. Here, we evaluated two different recycling Rab GTPases, Rab4a and Rab11a, in regulating endothelial VEGFR2 trafficking and signalling with implications for endothelial cell migration, proliferation and angiogenesis. In primary endothelial cells, VEGFR2 displays co-localisation with Rab4a, but not Rab11a GTPase, on early endosomes. Expression of a guanosine diphosphate (GDP)-bound Rab4a S22N mutant caused increased VEGFR2 accumulation in endosomes. TfR and VEGFR2 exhibited differences in endosome-to-plasma membrane recycling in the presence of chloroquine. Depletion of Rab4a, but not Rab11a, levels stimulated VEGF-A-dependent intracellular signalling. However, depletion of either Rab4a or Rab11a levels inhibited VEGF-A-stimulated endothelial cell migration. Interestingly, depletion of Rab4a levels stimulated VEGF-A-regulated endothelial cell proliferation. Rab4a and Rab11a were also both required for endothelial tubulogenesis. Evaluation of a transgenic zebrafish model showed that both Rab4 and Rab11a are functionally required for blood vessel formation and animal viability. Rab-dependent endosome-to-plasma membrane recycling of VEGFR2 is important for intracellular signalling, cell migration and proliferation during angiogenesis.

Understanding the molecular mechanisms and biological consequences of genetic changes occurring during bypass of cellular senescence spans a broad area of medical research from the cancer field to regenerative medicine. Senescence escape and immortalisation have been intensively studied in murine embryonic fibroblasts as a model system, and are known to occur when the p53/ARF tumour suppressor pathway is disrupted. We showed recently that murine fibroblasts with a humanised p53 gene (Hupki cells, from a human p53 knock-in mouse model) first senesce, and then become immortalised in the same way as their homologues with normal murine p53. In both cell types, immortalised cultures frequently sustain either a p53 gene mutation matching a human tumour mutation and resulting in loss of p53 transcriptional transactivation, or a biallelic deletion at the p19/ARF locus. Whilst these genetic events were not unexpected, we were surprised to find that a significant proportion of immortalised cell cultures apparently had neither a p53 mutation nor loss of p19/ARF. Here we consider various routes to p53/ARF disruption in senescence bypass, and dysfunction of other tumour suppressor networks that may contribute to release from tenacious cell cycle arrest in senescent cultures.

The regulated generation of prostaglandins from endothelial cells is critical to vascular function. Here we identify a novel mechanism for the regulation of endothelial cell prostaglandin generation. Cytosolic phospholipase A 2 -␣ (cPLA 2 ␣) cleaves phospholipids in a Ca 2؉ -dependent manner to yield free arachidonic acid and lysophospholipid. Arachidonic acid is then converted into prostaglandins by the action of cyclooxygenase enzymes and downstream synthases. By previously undefined mechanisms, nonconfluent endothelial cells generate greater levels of prostaglandins than confluent cells. Here we demonstrate that Ca 2؉-independent association of cPLA 2 ␣ with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA 2 ␣ and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA 2 ␣ is associated with the phospholipid-binding protein annexin A1. Furthermore, the siRNA-mediated knockdown of endogenous annexin A1 significantly reverses the inhibitory effect of confluence on endothelial cell prostaglandin generation. Thus the confluence-dependent interaction of cPLA 2 ␣ and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA 2 ␣ activity and endothelial cell prostaglandin generation.

Certain mutations within c-KIT cause constitutive activation of the receptor and have been associated with several human malignancies. These include gastrointestinal stromal tumors (GIST), mastocytosis, acute myelogenous leukemia, and germ cell tumors. The kinase inhibitor imatinib potently inhibits c-KIT and is approved for treatment of GIST. However, secondary point mutations can develop within the kinase domain to confer resistance to imatinib and cause drug-resistant relapse. A common mutation, which results in a V654A substitution,