Maria Radu

PAK signalling during the development and progression of cancer

p21-activated kinases (PAKs) are positioned at the nexus of several oncogenic signalling pathways. Overexpression or mutational activation of PAK isoforms frequently occurs in various human tumours, and recent data suggest that excessive PAK activity drives many of the cellular processes that are the hallmarks of cancer. In this Review, we discuss the mechanisms of PAK activation in cancer, the key substrates that mediate the developmental and oncogenic effects of this family of kinases, and how small-molecule inhibitors of these enzymes might be best developed and deployed for the treatment of cancer.

TGF-Beta Induced Erk Phosphorylation of Smad Linker Region Regulates Smad Signaling

The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

Alphaherpesvirus US3-mediated reorganization of the actin cytoskeleton is mediated by group A p21-activated kinases

The US3 protein is a viral serine/threonine kinase that is conserved among all members of the Alphaherpesvirinae. The US3 protein of different alphaherpesviruses causes dramatic alterations in the actin cytoskeleton, such as the disassembly of actin stress fibers and formation of cell projections, which have been associated with increased intercellular virus spread. Here, we find that inhibiting group A p21-activated kinases (PAKs), which are key regulators in Cdc42/Rac1 Rho GTPase signaling pathways, impairs US3-mediated actin alterations. By using PAK1−/− and PAK2−/− mouse embryo fibroblasts (MEFs), we show that US3-mediated stress fiber disassembly requires PAK2, whereas US3-mediated cell projection formation mainly is mediated by PAK1, also indicating that PAK1 and PAK2 can have different biological effects on the organization of the actin cytoskeleton. In addition, US3 was found to bind and phosphorylate group A PAKs. Lack of group A PAKs in MEFs was correlated with inefficient virus spread. Thus, US3 induces its effect on the actin cytoskeleton via group A PAKs.

The DeMSTification of Mammalian Ste20 Kinases

When first reported in 1995, the mammalian Ste20-like kinases (Mst) 1 and 2 were so named both for their similarity to the yeast kinase Ste20 and for the fact that their function was, to us, a deep mystery. While much remains to be explained about the regulation and role of these kinases, the veil has been at least partly raised on the Msts, revealing unexpected modes of activation and function. Work in model organisms suggests a central growth-suppressive role for Mst orthologs, with intriguing possible links to other established tumor suppressors. This minireview underlines our current understanding of how Mst1 and Mst2 are regulated, and how activation of these proteins influences cell survival and proliferation.

An emerging role for p21-activated kinases (Paks) in viral infections.

p21-activated protein kinases (Paks) are cytosolic serine/threonine protein kinases that act as effectors for small (p21) GTPases of the Cdc42 and Rac families. It has long been established that Paks play a major role in a host of vital cellular functions such as proliferation, survival and motility, and abnormal Pak function is associated with a number of human diseases. Here, we discuss emerging evidence that these enzymes also play a major role in the entry, replication and spread of many important pathogenic human viruses, including HIV. Careful assessment of the potential role of Paks in antiviral immunity will be pivotal to evaluate thoroughly the potential of agents that inhibit Pak as a new class of anti-viral therapeutics.

Pak2 Kinase Restrains Mast Cell FcϵRI Receptor Signaling through Modulation of Rho Protein Guanine Nucleotide Exchange Factor (GEF) Activity

Background: The protein kinase Pak1 stimulates mast cell degranulation, but the role of the more abundant isoform Pak2 in these cells is unknown. Results: Pak2, unlike Pak1, inhibits mast cell degranulation via a GEF-H1-RhoA pathway. Conclusion: Pak2 mediates signals between FcϵRI and secretion through regulation of the GEF responsible for RhoA activation. Significance: Pak2 has an opposing role to Pak1 in mast cell degranulation. p21-activated kinase-1 (Pak1) is a serine/threonine kinase that plays a key role in mediating antigen-stimulated extracellular calcium influx and degranulation in mast cells. Another isoform in this kinase family, Pak2, is expressed at very high levels in mast cells, but its function is unknown. Here we show that Pak2 loss in murine bone marrow-derived mast cells, unlike loss of Pak1, induces increased antigen-mediated adhesion, degranulation, and cytokine secretion without changes to extracellular calcium influx. This phenotype is associated with an increase in RhoA-GTPase signaling activity to downstream effectors, including myosin light chain and p38MAPK, and is reversed upon treatment with a Rho-specific inhibitor. Pak2, but not Pak1, negatively regulates RhoA via phosphorylation of the guanine nucleotide exchange factor GEF-H1 at an inhibitory site, leading to increased GEF-H1 microtubule binding and loss of RhoA stimulation. These data suggest that Pak2 plays a unique inhibitory role in mast cell degranulation by down-regulating RhoA via GEF-H1.

Pak2 restrains endomitosis during megakaryopoiesis and alters cytoskeleton organization

Megakaryocyte maturation and polyploidization are critical for platelet production; abnormalities in these processes are associated with myeloproliferative disorders, including thrombocytopenia. Megakaryocyte maturation signals through cascades that involve p21-activated kinase (Pak) function; however, the specific role for Pak kinases in megakaryocyte biology remains elusive. Here, we identify Pak2 as an essential effector of megakaryocyte maturation, polyploidization, and proplatelet formation. Genetic deletion of Pak2 in murine bone marrow is associated with macrothrombocytopenia, altered megakaryocyte ultrastructure, increased bone marrow megakaryocyte precursors, and an elevation of mature CD41(+) megakaryocytes, as well as an increased number of polyploid cells. In Pak2(-/-) mice, platelet clearance rate was increased, as was production of newly synthesized, reticulated platelets. In vitro, Pak2(-/-) megakaryocytes demonstrate increased polyploidization associated with alterations in β1-tubulin expression and organization, decreased proplatelet extensions, and reduced phosphorylation of the endomitosis regulators LIM domain kinase 1, cofilin, and Aurora A/B/C. Together, these data establish a novel role for Pak2 as an important regulator of megakaryopoiesis, polyploidization, and cytoskeletal dynamics in developing megakaryocytes.

Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

The molecular mechanisms that govern thymocyte development and maturation are incompletely understood. The P21-activated kinase 2 (Pak2) is an effector for the Rho family GTPases Rac and Cdc42 that regulate actin cytoskeletal remodeling, but its role in the immune system remains poorly understood. In this study, we show that T-cell specific deletion of Pak2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development, maturation, and egress of thymocytes. Pak2 was required for pre-TCR β-selection and positive selection. Surprisingly, Pak2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2. Mechanistically, Pak2 is required for actin cytoskeletal remodeling triggered by TCR. Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of Pak2, uncovering the critical function of Pak2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation. DOI: http://dx.doi.org/10.7554/eLife.02270.001

p21-Activated Kinase 2 Regulates Endothelial Development and Function through the Bmk1/Erk5 Pathway

ABSTRACT p21-activated kinases (Paks) have been shown to regulate cytoskeleton rearrangements, cell proliferation, attachment, and migration in a variety of cellular contexts, including endothelial cells. However, the role of endothelial Pak in embryo development has not been reported, and currently, there is no consensus on the endothelial function of individual Pak isoforms, in particular p21-activated kinase 2 (Pak2), the main Pak isoform expressed in endothelial cells. In this work, we employ genetic and molecular studies that show that Pak2, but not Pak1, is a critical mediator of development and maintenance of endothelial cell function. Endothelial depletion of Pak2 leads to early embryo lethality due to flawed blood vessel formation in the embryo body and yolk sac. In adult endothelial cells, Pak2 depletion leads to severe apoptosis and acute angiogenesis defects, and in adult mice, endothelial Pak2 deletion leads to increased vascular permeability. Furthermore, ubiquitous Pak2 deletion is lethal in adult mice. We show that many of these defects are mediated through a newly unveiled Pak2/Bmk1 pathway. Our results demonstrate that endothelial Pak2 is essential during embryogenesis and also for adult blood vessel maintenance, and they also pinpoint the Bmk1/Erk5 pathway as a critical mediator of endothelial Pak2 signaling.