Stephen Pak et al. (2001) International C. elegans Meeting "Emergence of an Ancient Intracellular Antiproteinase Defense System"

Stephen Pak, Yuko Sakaguchi, David Askew, Gary A Silverman, Chris Tsu, Cliff Luke, Sule Cataltepe, Vasantha Kumar

[International C. elegans Meeting, 2001]

Background: High molecular weight ser ine p roteinase in hibitor s (serpins) such as antithrombin III, a 2-antiplasmin and a 1-antitrypsin are critical regulators of intravascular (clotting and thrombolytic) and extravascular proteolytic cascades. We have characterized a family (n>13) of human ovalbumin–like Serpins. Unlike the fluid phase serpins, the ov-serpins lack N-terminal signal peptides and reside within the cytosol of epithelial cells. However, the function of these molecules is unknown. Objective: Determine whether the function of the ov-serpins can be elucidated using a comparative genomics approach and a simpler organism, C. elegans . Design/Methods: Database analysis and molecular cloning were used to identify serpins. Results: Screening of ACeDB revealed the presence of 9-10 functional serpin genes ( srp-1-10 ). They are tandemly arrayed along Chromosome V. By a combination of cDNA cloning and RT-PCR, cDNAs corresponding to 6 of the genes were isolated. The genes contain 5-6 exons with conserved splice sites at the 5' and 3' ends but some variation within. Like the ov-serpins, none of the worm serpins encode for typical N-terminal signal peptides. Thus, C.elegans serpins also are likely to reside intracellularly. Each serpin contains different residues in the reactive site loop (the reactive site loop is that portion of the serpin that binds to the active site of the proteinase). This variation suggests that each serpin will inhibit different types of proteinases. Indeed, kinetic analysis in vitro, using recombinant proteins expressed in E.coli , showed that srp-2 and srp-3 are inhibitors ( k ass ~1x10 4 M -1 sec -1 ) of granzyme B and chymotrypsin-like serine proteinases, respectively. Preliminary transgenic studies using a vector containing the srp-2 promotor fused to a GFP reporter gene showed that, like the ov-serpins, srp-2 is highly expressed in the hypoderm. Conclusions: Members of the ov-serpin family are functionally and topologically conserved in C.elegans . Reverse genetic studies by the generation of null mutations should reveal the functions of this intracellular anti-proteinase defense system that is highly conserved through out metazoan evolution.

Stephen C Pak et al. (2004) East Coast Worm Meeting "SRP-2 is an intracellular serpin that participates in postembryonic development"

Anthony C Clark, David R Mills, David J Askew, Christopher Tsu, Stephen C Pak, Vasantha Kumar, Gary Silverman, Yuko S Askew, Cliff J Luke

[East Coast Worm Meeting, 2004]

Serpins are high molecular weight serine proteinase inhibitors that inactivate target proteinases by a suicide substrate-like mechanism. Serpins can be categorized into two broad groups, intracellular and extracellular. Most extracellular serpins function in the circulation and regulate proteinases involved in blood coagulation, fibrinolysis, complement activation, inflammation, and extracellular matrix remodeling. In contrast, serpins belonging to the intracellular group have been implicated in regulating apoptosis, tumor progression, and metastasis. However, their functions in terms of an intact organism have not been well defined. Database analysis of the C. elegans genome reveals the presence of several intracellular serpins. To determine whether nematode serpins function as proteinase inhibitors, one family member, srp-2 , was chosen for further characterization. Biochemical analysis of recombinant SRP-2 protein revealed SRP-2 to be a potent inhibitor of the lysosomal cysteine proteinases, cathepsins K, L, S, and V. Analysis of temporal and spatial expression indicated that SRP-2 was present during early embryonic development and highly expressed in the hypoderm of larval and adult worms. To determine whether SRP-2 plays a role in C. elegans development, null mutants and transgenic animals overexpressing SRP-2 were generated. Whereas null mutants showed no overt developmental phenotype, of the animals overexpressing SRP-2 ~30% displayed developmental abnormalities characterized by early (L1/L2) larval arrest/death, slow growth and molting defects at 25 degC. However, at 27 degC the phenotype was considerably more penetrant with ~95% of the animals being abnormal. Although the mode of SRP-2 action is currently unknown, we hypothesize that SRP-2 plays a role in regulating proteinase activity during development and that an imbalance in the serpin/proteinase equilibrium has deleterious consequences during C. elegans development.

Stephen C Pak et al. (2002) East Coast Worm Meeting "SERPIN FUNCTION IN C. ELEGANS: A GENETIC AND BIOCHEMICAL ANALYSIS"

Cliff J Luke, Gary A Silverman, David J Askew, Yuko S Askew, David R Mills, Stephen C Pak, Sule Cataltepe, Christopher Tsu, Vasantha Kumar

[East Coast Worm Meeting, 2002]

To better understand the biology of ov-serpins, we undertook a comparative genomics study using C. elegans as a model organism. A screen of the C. elegans database (ACeDB ) using the human serpin, SCCA2, amino acid sequence identified the presence of 9-10 serpin genes (srp-1-10). These genes are arranged in tandem and are distributed throughout the length of chromosome V. By a combination of cDNA cloning and RT-PCR, cDNAs corresponding to 6 of these genes were isolated. Like the human ov-serpins, no typical N-terminal signal peptides were found suggesting that the worm serpins may also reside intracellularly. Each serpin contains different residues in the reactive site loop suggesting that they have distinct proteinase targets. Interestingly and reminiscent of the Manduca sexta serpin gene-1, srp-7 was found to have 3 distinct reactive site loops resulting from alternative splicing of exon 5. In vitro kinetic analysis, using recombinant proteins expressed in E. coli, showed that srp-2, srp-3 and srp-6 are inhibitors (kass _1x104 M-1 sec-1 ) of granzyme B, chymotrypsin-like serine proteinases, and papain-like cysteine proteinases, respectively. Expression studies using promoter::GFP-fusions indicate that serpins are present in the hypoderm, intestine, neurons and excretory glands. Null deletion mutants of srp-1, srp-2, srp-6, srp-7(a-c) and srp-8 have been isolated and initial characterizations suggest that serpins play important roles in osmoregulation and host defense against bacterial infection.

Stephen C. Pak et al. (2009) International Worm Meeting "An automated high-content screen for drugs that alleviate misfolded protein aggregation."

David H. Perlmutter, Gary A. Silverman, Sager Gosai, Olivia Long, Stephen C. Pak

[International Worm Meeting, 2009]

Alpha-1-antitrypsin (AT) deficiency is member of a growing family of human genetic disorders (e.g., Huntingtons and Alzheimers) called "conformational" diseases. These diseases are characterized by protein misfolding, polymerization and/or aggregation. In the case of AT-deficiency, the accumulation of abnormal proteins within hepatocytes leads to liver injury and degenerative disease. To facilitate identification of therapeutic compounds, we have developed an automated, 384-well-based, high-content drug screen using the ArrayScan VTi imaging platform. A pilot screen of the LOPAC library identified 12 hit compounds. Of these, one compound has already been reported to have an effect on AT accumulation. Further characterization of other hit compounds should shed light on drugs that alleviate cellular toxicity associated with protein aggregation. This method can be easily adapted to other C. elegans models and provides a stepping-stone for future C. elegans-based drug screens.

Stephen C Pak et al. (2005) International Worm Meeting "Protein aggregate formation and disruption of post-embryonic development in worms expressing SRP-2::GFP"

Clifford J Luke, Stephen C Pak, Gary A Silverman

[International Worm Meeting, 2005]

Serpins are high molecular weight serine proteinase inhibitors that irreversibly inactivate target proteinases using a suicide substrate-like mechanism. Although in vitro biochemical characterizations have provided some clues as to the identity of the target proteinases, the pathways in which serpins play a role are currently unknown. To determine whether serpins plays a role in C. elegans development, growth and longevity, we generated mutants that lack or over-express serpin genes. In general, with the exception of srp-6 (see poster by Luke et al.,), null mutants displayed no overt phenotypes. In contrast, transgenic animals over-expressing serpins displayed a variety of developmental and growth defects. In particular, over-expression of SRP-2::GFP resulted in distruption of post-embryonic development characterized by early larval arrest, slow growth and molting defects. Over-expression of SRP-2 mutants harboring amino-acid substitutions that diminish the serpins ability to inhibit proteinases failed to suppress the phenotype. This result suggests that the phenotype is independent of the proteinase-inhibitory function of SRP-2. Preliminary structure/function mapping results suggest that a short, N-terminal portion of SRP-2 is sufficient for the phenotype. Interestingly, we observed large, insoluble SRP-2::GFP aggregates in the cytosol of anterior hypodermal cells. The relationship between aggregate formation and the developmental defects is currently unclear. We are currently conducting a genetic screen to identify modifiers of the SRP-2 developmental and aggregation phenotypes. We hope to present the results of the genetic screen at the conference.

Peters EC et al. (2013) G3 (Bethesda) "Redundant canonical and noncanonical Caenorhabditis elegans p21-activated kinase signaling governs distal tip cell migrations."

Gossett AJ, Peters EC, Reiner DJ, Goldstein B, Der CJ

[G3 (Bethesda), 2013]

p21-activated kinases (Paks) are prominent mediators of Rac/Cdc42-dependent and -independent signaling and regulate signal transduction and cytoskeletal-based cell movements. We used the reproducible migrations of the Caenorhabditis elegans gonadal distal tip cells to show that two of the three nematode Pak proteins, MAX-2 and PAK-1, function redundantly in regulation of cell migration but are regulated by very different mechanisms. First, we suggest that MAX-2 requires CED-10/Rac function and thus functions canonically. Second, PIX-1 and GIT-1 function in the same role as PAK-1, and PAK-1 interaction with PIX-1 is required for PAK-1 activity; thus, PAK-1 functions noncanonically. The human Pak-Pix-Git complex is central to noncanonical Pak signaling and requires only modest Rac/CDC-42 input. Unlike the human complex, our results suggest that the C. elegans Pak-Pix-Git complex requires PAK-1 kinase domain activity. This study delineates signaling network relationships in this cell migration model, thus providing potential further mechanistic insights and an assessment of total Pak contribution to cell migration events.

Lucanic M et al. (2008) PLoS Genet "A RAC/CDC-42-independent GIT/PIX/PAK signaling pathway mediates cell migration in C. elegans."

Lucanic M, Cheng HJ

[PLoS Genet, 2008]

P21 activated kinase (PAK), PAK interacting exchange factor (PIX), and G protein coupled receptor kinase interactor (GIT) compose a highly conserved signaling module controlling cell migrations, immune system signaling, and the formation of the mammalian nervous system. Traditionally, this signaling module is thought to facilitate the function of RAC and CDC-42 GTPases by allowing for the recruitment of a GTPase effector (PAK), a GTPase activator (PIX), and a scaffolding protein (GIT) as a regulated signaling unit to specific subcellular locations. Instead, we report here that this signaling module functions independently of RAC/CDC-42 GTPases in vivo to control the cell shape and migration of the distal tip cells (DTCs) during morphogenesis of the Caenorhabditis elegans gonad. In addition, this RAC/CDC-42-independent PAK pathway functions in parallel to a classical GTPase/PAK pathway to control the guidance aspect of DTC migration. Among the C. elegans PAKs, only PAK-1 functions in the GIT/PIX/PAK pathway independently of RAC/CDC42 GTPases, while both PAK-1 and MAX-2 are redundantly utilized in the GTPase/PAK pathway. Both RAC/CDC42-dependent and -independent PAK pathways function with the integrin receptors, suggesting that signaling through integrins can control the morphology, movement, and guidance of DTC through discrete pathways. Collectively, our results define a new signaling capacity for the GIT/PIX/PAK module that is likely to be conserved in vertebrates and demonstrate that PAK family members, which are redundantly utilized as GTPase effectors, can act non-redundantly in pathways independent of these GTPases.

Chamberlain SH et al. (1994) Worm Breeder's Gazette "A Molecular Approach to Identify skn-1-Responsive Genes in the Early C. elegans Embryo"

Chamberlain SH, Bowerman BA

[Worm Breeder's Gazette, 1994]

A molecular approach to identify skn-1-responsive genes in the early C. elegans embryo. Stephen H. Chamberlain and Bruce Bowerman, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403

Goh KY et al. (2012) Proc Natl Acad Sci U S A "p21-activated kinase interacts with Wnt signaling to regulate tissue polarity and gene expression."

Ng NW, Inoue T, Hagen T, Goh KY

[Proc Natl Acad Sci U S A, 2012]

Wnt signaling is mediated by three classes of receptors, Frizzled, Ryk, and Ror. In Caenorhabditis elegans, Wnt signaling regulates the anterior/posterior polarity of the P7.p vulval lineage, and mutations in lin-17/Frizzled cause loss or reversal of P7.p lineage polarity. We found that pak-1/Pak (p21-activated kinase), along with putative activators of Pak, nck-1/Nck, and ced-10/Rac, regulates P7.p polarity. Mutations in these genes suppress the polarity defect of lin-17 mutants. Furthermore, mutations in pak-1, nck-1, and ced-10 cause constitutive dauer formation at 27 C, a phenotype also observed in egl-20/Wnt and cam-1/Ror mutants. In HEK293T cells, Pak1 can antagonize canonical Wnt signaling. Moreover, overexpression of Ror2 leads to phosphorylation of Pak1. Together, these results indicate that Pak interacts with Wnt signaling to regulate tissue polarity and gene expression.

Harel, S. et al. (2009) International Worm Meeting "git-1, pix-1 and pak-1 control embryo elongation through induction of myosin light chain phosphorylation in Caenorhabditis elegans."

Harel, S., Jenna, S., Lee, A.Y.

[International Worm Meeting, 2009]

git-1, pix-1 and pak-1 are the orthologs of human GIT (G protein-coupled receptor kinase interactor), PIX (Pak1-interacting exchange factor) and PAK (p21 activated kinase). GIT1 is a GAP (GTPase-activating protein) for the Arf GTPases. PIX is a GEF (guanine-nucleotide exchange factor) for the Rho GTPases Rac and Cdc42 and PAK an effector of these later GTPases. These proteins form a complex shown to control morphology and migration of neuronal and non-neuronal cells in vertebrates mainly through regulation of membrane trafficking, focal adhesion dynamics and actin-myosin-mediated contraction. In C. elegans, GIT/PIX/PAK complex together with the integrins were shown to control cell shape and migration of distal tip cells during gonad morphogenesis. We demonstrate here that git-1, pix-1 and pak-1 control early elongation events during C. elegans embryonic development. Early elongation involves machineries controlling the phosphorylation of the myosin-light chain (MLC), mlc-4, and consequently the contraction of the actin-myosin contractile apparatus (AMCA). The Rho-binding kinase let-502 was shown to promote AMCA contraction through inhibition of the MLC phosphatase activity of mel-11 in lateral epidermal seam cells. let-502/mel-11 elongation pathway was shown to act redundantly with a unknown pathway involving a MLC kinase activity. Interestingly, this parallel pathway involves fem-2, a serine-threonine phosphatase whose closer homolog in mammals PP2c is known to control PAK activity downstream of PIX. Using loss-of function and dominant negative alleles of pix-1, git-1 and pak-1, RNAi treatment, specific chemical inhibitors of MLC phosphorylation regulators and 4D-imaging we showed that reduction or suppression of GIT/PIX/PAK function results to similar elongation defects than observed in let-502 loss-of-function mutants. We also showed that git-1, pix-1 and pak-1 are antagonizing MLC dephosphorylation during elongation. Preliminary data suggest that this function of GIT/PIX/PAK during elongation would involve integrins. We are currently investigating whether this novel pathway acts in parallel to mel-11 and let-502 and if it involves fem-2.