Functional Deficits in Pak5, Pak6 and Pak5/Pak6 Knockout Mice

The p21-activated kinases are effector proteins for Rho-family GTPases. PAK4, PAK5, and PAK6 are the group II PAKs associated with neurite outgrowth, filopodia formation, and cell survival. Pak4 knockout mice are embryonic lethal, while Pak5, Pak6, and Pak5/Pak6 double knockout mice are viable and fertile. Our previous work found that the double knockout mice exhibit locomotor changes and learning and memory deficits. We also found some differences with Pak5 and Pak6 single knockout mice and the present work further explores the potential differences of the Pak5 knockout and Pak6 knockout mice in comparison with wild type mice. The Pak6 knockout mice were found to weigh significantly more than the other genotypes. The double knockout mice were found to be less active than the other genotypes. The Pak5 knockout mice and the double knockout mice performed worse on the rotorod test. All the knockout genotypes were found to be less aggressive in the resident intruder paradigm. The double knockout mice were, once again, found to perform worse in the active avoidance assay. These results indicate, that although some behavioral differences are seen in the Pak5 and Pak6 single knockout mice, the double knockout mice exhibit the greatest changes in locomotion and learning and memory.     

Targeted disruption of the Pak5 and Pak6 genes in mice leads to deficits in learning and locomotion

PAK6 is a member of the group B family of PAK serine/threonine kinases, and is highly expressed in the brain. The group B PAKs, including PAK4, PAK5, and PAK6, were first identified as effector proteins for the Rho GTPase Cdc42. They have important roles in filopodia formation, the extension of neurons, and cell survival. Pak4 knockout mice die in utero, and the embryos have several abnormalities, including a defect in the development of motor neurons. In contrast, Pak5 knockout mice do not have any noticeable abnormalities. So far nothing is known about the biological function of Pak6. To address this, we have deleted the Pak6 gene in mice. Since Pak6 and Pak5 are both expressed in the brain, we also generated Pak5/Pak6 double knockout mice. These mice were viable and fertile, but had several locomotor and behavioral deficits. Our results indicate that Pak5 and Pak6 together are not required for viability, but are required for a normal level of locomotion and activity as well as for learning and memory. This is consistent with a role for the group B PAKs in the nervous system.     

Pak functions downstream of Dock to regulate photoreceptor axon guidance in Drosophila.

The SH2/SH3 adaptor protein Dock has been proposed to transduce signals from guidance receptors to the actin cytoskeleton in Drosophila photoreceptor (R cell) growth cones. Here, we demonstrate that Drosophila p21-activated kinase (Pak) is required in a Dock pathway regulating R cell axon guidance and targeting. Dock and Pak colocalize to R cell axons and growth cones, physically interact, and their loss-of-function phenotypes are indistinguishable. Normal patterns of R cell connectivity require Pak's kinase activity and binding sites for both Dock and Cdc42/Rac. A membrane-tethered form of Pak (Pak(myr) acts as a dominant gain-of-function protein. Retinal expression of Pak(myr) rescues the R cell connectivity phenotype in dock mutants. These data establish Pak as a critical regulator of axon guidance and a downstream effector of Dock in vivo.     

Pak1 and Pak2 mediate tumor cell invasion through distinct signaling mechanisms

Pak kinases are thought to play critical roles in cell migration and invasion. Here, we analyze the roles of Pak1 and Pak2 in breast carcinoma cell invasion using the transient transfection of small interfering RNA. We find that although both Pak1 and Pak2 contribute to breast carcinoma invasion stimulated by heregulin, these roles are mediated by distinct signaling mechanisms. Thus, whereas the depletion of Pak1 interferes with the heregulin-mediated dephosphorylation of cofilin, the depletion of Pak2 does not. The depletion of Pak1 also has a stronger inhibitory effect on lamellipodial protrusion than does the depletion of Pak2. Interestingly, Pak1 and Pak2 play opposite roles in regulating the phosphorylation of the myosin light chain (MLC). Whereas the depletion of Pak1 decreases phospho-MLC levels in heregulin-stimulated cells, the depletion of Pak2 enhances MLC phosphorylation. Consistent with their opposite effects on MLC phosphorylation, Pak1 and Pak2 differentially modulate focal adhesions. Pak2-depleted cells display an increase in focal adhesion size, whereas in Pak1-depleted cells, focal adhesions fail to mature. We also found that the depletion of Pak2, but not Pak1, enhances RhoA activity and that the inhibition of RhoA signaling in Pak2-depleted cells decreases MLC phosphorylation and restores cell invasion. In summary, this work presents the first comprehensive analysis of functional differences between the Pak1 and Pak2 isoforms.     

Coriaria duthiei sp. nov. (Coriariaceae) from the Indo–Pak region

A new species of Coriaria L. (Coriariaceae), C. duthiei D. K. Singh & Pusalkar is described and illustrated from the Indo–Pak region. A key to the species of the genus and their distribution is provided.     

Failla Memorial Lecture. From beans to genes--back to the future.

Developments in radiation biology have inevitably paralleled the evolution of systems and end points in the wider field of biology. I review the development of three areas that have interested me during the 35 years that I have been involved with radiation research. (1) The dose-rate effect. My first graduate student, Joel Bedford, and I demonstrated the dependence of mammalian cell killing on the dose rate at which gamma rays are delivered. These studies led to the recent development of pulsed low-dose-rate brachytherapy and the design of a new machine for clinical use. (2) Hypoxic radiosensitizers and bioreductive drugs. The hypothesis that the presence of foci of hypoxic cells in human tumors could limit their curability by gamma rays led to the development of nitromidazales that preferentially sensitize hypoxic cells. Our contributions included the observation that misonidazole was also cytotoxic to hypoxic cells, that sensitization was increased by prolonged preincubation, and that the mechanism involved depletion of thiols. More recently we have collaborated with Ged Adams and Bob Sutherland in the development of a new generation of hypoxic cell cytotoxins. In particular, we have been concerned with the assessment of oncogenicity in relation to drug structure. (3) Oncogenic transformation in vitro. The use of in vitro assays for oncogenic transformation has represented a major interest for this laboratory largely due to collaborations first with Carmia Borek, and more recently with Richard Miller and Tom Hei. We published the first dose-response curves for gamma rays, and more recently for a range of neutron energies and for charged particles of defined LET. Radiation-induced oncogenic transformation in C3H 10T1/2 cells has been shown to be due to a dominant acting transforming gene, which has been isolated and is in the process of being characterized and sequenced by Greg Freyer.     

Clathrin-independent endocytosis used by the IL-2 receptor is regulated by Rac1, Pak1 and Pak2

There are several endocytic pathways, which are either dependent on or independent of clathrin. This study focuses on a poorly characterized mechanism-clathrin- and caveolae-independent endocytosis-used by the interleukin-2 receptor beta (IL-2R beta). We address the question of its regulation in comparison with the clathrin-dependent pathway. First, we show that Ras-related C3 botulinum toxin substrate 1 (Rac1) is specifically required for IL-2R beta entry, and we identify p21-activated kinases (Paks) as downstream targets. By RNA interference, we show that Pak1 and Pak2 are both necessary for IL-2R beta uptake, in contrast to the clathrin-dependent route. We observe that cortactin, a partner of actin and dynamin-two essential endocytic factors-is required for IL-2R beta uptake. Furthermore, we find that cortactin acts downstream from Paks, suggesting control of its function by these kinases. Thus, we describe a cascade composed of Rac1, Paks and cortactin specifically regulating IL-2R beta internalization. This study indicates Paks as the first specific regulators of the clathrin-independent endocytosis pathway.     

The Akt proto-oncogene links Ras to Pak and cell survival signals

The Ras oncogene regulates cellular proliferation, differentiation, transformation, and survival through multiple downstream signals. Ras signals through its effector phosphoinositide 3 (PI3) kinase to the Pak protein kinase (p65(pak)), but the steps from Ras to Pak remain to be elucidated. PI3 kinase can stimulate the small G protein, Rac, a direct activator of Pak, as well as the Akt proto-oncogene, a serine-threonine protein kinase. We found that activated Akt stimulated Pak, whereas a dominant negative Akt inhibited Ras activation of Pak in transfection assays. Akt stimulation of Pak was not inhibited by dominant negative mutants of either Rac or Cdc42 suggesting that Akt activated Pak through a GTPase-independent mechanism. We also developed a novel cell-free system to study Ras activation of Pak. In this system Ras activated Pak only in the presence of a crude cell extract but failed to activate Pak when Akt was immunodepleted from the extract. Akt protects cells from apoptosis through phosphorylation of downstream targets such as the Bcl-2 family member, Bad. We found that activated Pak decreased apoptosis and increased phosphorylation of Bad, whereas dominant negative Pak increased apoptosis and decreased phosphorylation of Bad. These studies define a new oncogene-mediated cell survival signal.     

cGMP-dependent protein kinase phosphorylates p21-activated kinase (Pak) 1, inhibiting Pak/Nck binding and stimulating Pak/vasodilator-stimulated phosphoprotein association

Endothelial cells are normally non-motile and quiescent; however, endothelial cells will become permeable and invade and proliferate to form new blood vessels (angiogenesis) in response to wounding, cancer, diabetic retinopathy, age-related macular degeneration, or rheumatoid arthritis. p21-activated kinase (Pak), an effector for the Rho GTPases Rac and Cdc42, is required for angiogenesis and regulates endothelial cell permeability and motility. Although Pak is primarily activated by Rac and Cdc42, there are additional proteins that regulate Pak activity and localization, including three AGC protein kinase family members, Akt-1, PDK-1, and cAMP-dependent protein kinase. We describe phosphorylation and regulation of Pak localization by a fourth AGC kinase family member, cGMP-dependent protein kinase (PKG). Using in vitro mapping, a phosphospecific antibody, co-transfection assays, and untransfected bovine aortic endothelial cells we determined that PKG phosphorylates Pak at serine 21. Phosphorylation was accompanied by changes in proteins associated with Pak. The adaptor protein Nck was released, whereas a novel complex with vasodilator-stimulated phosphoprotein was stimulated. Furthermore Ser-21 phosphorylation of Pak appears to be important for regulation of cell morphology. In both human umbilical vein endothelial cells and HeLa cells, activation of PKG in the presence of Pak stimulated tail retraction and cell polarization. However, in cells expressing S21A mutant Pak1, PKG activation or treatment with a peptide that blocks Nck/Pak binding caused aberrant cell morphology, blocked cell retraction, and mislocalized Pak, producing uropod (tail-like) structures. These data suggest that PKG regulates Pak and that the interaction plays a role in tail retraction.     

Lentivirus Nef specifically activates Pak2

Nef proteins from human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) have been found to associate with an active cellular serine/threonine kinase designated Nef-associated kinase (Nak). The exact identity of Nak remains controversial, with two recent studies indicating that Nak may be either Pak1 or Pak2. In this study, we investigated the hypothesis that such discrepancies arise from the use of different Nef alleles or different cell types by individual investigators. We first confirm that Pak2 but not Pak1 is cleaved by caspase 3 in vitro and then demonstrate that Nak is caspase 3 sensitive, regardless of Nef allele or cell type used. We tested nef alleles from three lentiviruses (HIV-1 SF2, HIV-1 NL4-3, and SIVmac239) and used multiple cell lines of myeloid, lymphoid, and nonhematopoietic origin to evaluate the identity of Nak. We demonstrate that ectopically expressed Pak2 can substitute for Nak, while ectopically expressed Pak1 cannot. We then show that Nef specifically mediates the robust activation of ectopically expressed Pak2, directly demonstrating that Nef regulates Pak2 activity and does not merely associate with activated Pak2. We report that most of the active Pak2 is found bound to Nef, although a fraction is not. In contrast, only a small amount of Nef is found associated with Pak2. We conclude that Nak is Pak2 and that Nef specifically mediates Pak2 activation in a low-abundance complex. These results will facilitate both the elucidation of the role of Nef in pathogenesis and the development of specific inhibitors of this highly conserved function of Nef.     

Microbiologically influenced corrosion: looking to the future.

This review discusses the state-of-the-art of research into biocorrosion and the biofouling of metals and alloys of industrial usage. The key concepts needed to understand the main effects of microorganisms on metal decay, and current trends in monitoring and control strategies to mitigate the deleterious effects of biocorrosion and biofouling are also described. Several relevant cases of biocorrosion studied by our research group are provided as examples: (i) biocorrosion of aluminum and its alloys by fungal contaminants of jet fuels; (ii) sulfate-reducing bacteria (SRB)-induced corrosion of steel; (iii) biocorrosion and biofouling interactions in the marine environment; (iv) monitoring strategies for assessing biocorrosion in industrial water systems; (v) microbial inhibition of corrosion; (vi) use and limitations of electrochemical techniques for evaluating biocorrosion effects. Future prospects in the field are described with respect to the potential of innovative techniques in microscopy (environmental scanning electron microscopy, confocal scanning laser microscopy, atomic force microscopy), new spectroscopic techniques for the study of corrosion products and biofilms (energy dispersion X-ray analysis, X-ray photoelectron spectroscopy, electron microprobe analysis) and electrochemistry (electrochemical impedance spectroscopy, electrochemical noise analysis).     

Pondering the future.

Despite growing tensions as the Canadian health care system evolves, the system will survive over the next decade and strengthen its societal objectives, sometimes dragging physicians along. With the shift of the intellectual centre away from the universities, research and researchers will find funding security but career insecurity in the private sector. The independent investigator will become rare as "big science" becomes the norm, and basic science departments will have a decade of confusion as they struggle for a new place in the renewed medical schools. The move toward problem-based, community-oriented medical education will be completed, clinical faculty will become salaried and the smaller numbers of graduates will be more controlled in their practices. Medicine as a profession will adopt a renewed philosophical framework and will broaden in its scope to the benefit of Canadians.     

Kinase-deficient Pak1 mutants inhibit Ras transformation of Rat-1 fibroblasts.

Among the mechanisms by which the Ras oncogene induces cellular transformation, Ras activates the mitogen-activated protein kinase (MAPK or ERK) cascade and a related cascade leading to activation of Jun kinase (JNK or SAPK). JNK is additionally regulated by the Ras-related G proteins Rac and Cdc42. Ras also regulates the actin cytoskeleton through an incompletely elucidated Rac-dependent mechanism. A candidate for the physiological effector for both JNK and actin regulation by Rac and Cdc42 is the serine/threonine kinase Pak (p65pak). We show here that expression of a catalytically inactive mutant Pak, Pak1(R299), inhibits Ras transformation of Rat-1 fibroblasts but not of NIH 3T3 cells. Typically, 90 to 95% fewer transformed colonies were observed in cotransfection assays with Rat-1 cells. Pak1(R299) did not inhibit transformation by the Raf oncogene, indicating that inhibition was specific for Ras. Furthermore, Rat-1 cell lines expressing Pak1(R299) were highly resistant to Ras transformation, while cells expressing wild-type Pak1 were efficiently transformed by Ras. Pak1(L83,L86,R299), a mutant that fails to bind either Rac or Cdc42, also inhibited Ras transformation. Rac and Ras activation of JNK was inhibited by Pak1(R299) but not by Pak1(L83,L86,R299). Ras activation of ERK was inhibited by both Pak1(R299) and Pak1(L83,L86,R299), while neither mutant inhibited Raf activation of ERK. These results suggest that Pak1 interacts with components essential for Ras transformation and that inhibition can be uncoupled from JNK but not ERK signaling.