ROLE OF P21-ACTIVATED KINASE (PAK)-NCK BINDING IN THE FORMATION OF FILOPODIA AND LARGE PROTRUSIONS John Gary DeMuth A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of Master of Science May 2010 Committee: Carol A. Heckman, Advisor Michael E. Geusz Mikhail A. Zamkov ii ABSTRACT Carol Heckman, Advisor The p21-activated kinases (PAK) isoforms 1-3 are serine-threonine kinases [1]. One potential binding partner for PAK is the non-catalytic tyrosine kinase adaptor protein, Nck [2]. It had previously been found that large protrusions decreased in rat 1000 W respiratory tract epithelial cells when the Nck-binding portion of PAK 1 (PAK7-24) was introduced into them. The cell line used was an in vitro model of lung cancer. When the corresponding Nck SH3[2] domain was introduced, the same was not observed. It was also found that the Nck-binding segment of PAK 1, PAK7-24, had no effect upon filopodia, while introduction of the PAK-binding Nck SH3[2] domain decreased filopodia formation. One possible interpretation for the results was that, for each protein, there was another partner (not Nck or PAK) binding at the same domain. Another explanation for the observations was that two PAK isoforms were required, one for each type of protrusion [3]. To determine if different PAK isoforms were involved in filopodia and large protrusion formation, the 1-25 N-terminal amino acid sequence of each PAK isoform was introduced. This was preceded in some treatments by introduction of siRNAs against PAK 1-3 isoforms separately to knock down transcript production. Cells were then prepared for viewing by scanning electron microscopy. Micrographs of random cells were taken, their edges traced, and these tracings then scanned and transferred to a computer program that analyzed cell shape. Differences between sample means were determined by analysis of variance (ANOVA), and Duncan's multiple range test was employed to evaluate the relationship among samples. Only PAK 2 was associated with filopodia formation. Specific PAK 2 siRNA inhibited filopodia, while the PAK 21-25 peptide reversed the inhibition of these structures by tumor iii promoters. Specific siRNA against PAK 1 and PAK 2 were each found to block large protrusion formation. However, this only decreased the prevalence of large protrusions to the level of the sham-treated and untreated control samples, because the respective 1-25 N-terminal sequences had the unexpected effect of enhancing large protrusion formation compared controls. The results supported isoform-specific formation of the protrusions. iv ACKNOWLEDGEMENTS I would like to thank the late Dr. Stan Smith for aiding my entry into the Biological Sciences graduate program at Bowling Green State University. I would like to thank Dr. Marilyn Cayer for her direction in my utilization of various machines of the electron microscopy center, as well as the many cell tracings she graciously completed. I would like to thank Dr. Nancy Boudreau and her various graduate students for their aid in analyzing and interpreting my data. I would like to thank Patrick Richey, Deirdre Dobos, and Santosh Malwade for their efforts in tracing cell micrographs used in my thesis experiment. I would like to thank Drs. Michael Geusz and Mikhail Zamkov for their roles as members of my thesis committee. And I would especially like to thank Dr. Carol Heckman for her patient, tireless support and intelligent guidance while I was an undergraduate and as a Master’s student in her laboratory. To all, I will be forever grateful, striving to help others as you have me. v TABLE OF CONTENTS INTRODUCTION……..……..…………………………………………………….. 1 MATERIALS AND METHODS……..…….………………………..……………… 9 RESULTS………………...…………………………………………………………. 15 DISCUSSION………………………………………………………………………. 21 REFERENCES…………………………………………………………………….... 24 APPENDIX…………………………………………………………………………. 43 vi TABLES TEXT TABLES 6 Factor #4 values after PAK knockdown and PAK peptide interference…………. 16 7 Factor #7 values after PAK knockdown and PAK peptide interference………….. 19 8 Selected factor 7 values after PAK knockdown and PAK peptide interference….. 20 APPENDIX TABLES 1 Upstream effectors of Rac- or Cdc42-mediated PAK activation …………………. 43 2 Effectors of PAK activation not requiring GTPases Rac or Cdc42………………. 44 3 Effectors of PAK inactivation……………………………………………………... 45 4 Binding partners of Nck's SH2 domain…………………………………………… 46 5 Binding partners of Nck's SH3 domains…………………………………………... 47 vii FIGURES 1 Schematic diagram indicating features of PAK1 structure…….……………..…... 4 2 Schematic diagram of PAK1 autoregulation and activation by Cdc42………….. 5 3 Modular composition of Nck adaptor proteins…………………………………..... 6 4 Hypothesis of isoform-selective Nck-PAK binding in protrusion formation…….. 7 5 Example of a 1000 W cell as observed by scanning electron microscopy………… 13 1 INTRODUCTION Embryonic development, tissue repair, and immune system functioning, as well as the pathological processes of inflammatory disease and tumor cell invasion, all depend upon cell migration [4-6]. A cell's migration can be characterized by its speed and directionality, which are the manifestations of and dependent upon the complex interactions among protrusion formation, membrane interactions with the substrate, and cytoskeletal dynamics migration [4, 5, 7, 8]. Directed migration can be differentiated by whether it is controlled by intrinsic or external cues [9]. The former refers to “the propensity of cells to continue migrating in the same direction without turning”, while the latter refers to the process of chemotaxis [9]. Inducing genetic mutations to PAK and dreadlocks (dock), a Nck homolog, showed the two to be essential in the oriented movement of Drosophila melanogaster photoreceptor axon guidance [10], with dock appearing to target PAK to focal contacts during this process [11]. Examining whether the same relationship of Nck and PAK, with regards to persistent directional movement, exists in rat epithelial respiratory cells is the focus of the current research. Actin, focal contacts, and cellular protrusions Actin filaments, along with intermediate filaments and microtubules, comprise the main structural components of the eukaryotic cytoskeleton. Connecting the actin to the extracellular matrix are integrin-mediated structures termed focal contacts [12]. The adaptor protein, paxillin, has a primary role in the formation and dissolution of these focal contacts [13]. It is proposed that, upon its phosphorylation by a kinase downstream of a GTPase, cell division cycle 42 (Cdc42) or Ras-related C3 botulinum toxin substrate 1 (Rac), paxillin is then able to interact with G-protein coupled receptor kinase interacting proteins, GIT1 and GIT2. GIT1 is bound, through 2 its Spa2 homology domain, to PAK-interacting exchange factor protein (PIX). Phosphorylation of paxillin Ser-273 by PAK appears to leave GIT1 binding unaffected while removing the site where focal adhesion kinase (FAK) binds to paxillin [14]. The turnover of the focal contacts involves similar mechanisms [15-17]. One type of actin-based cellular protrusion is filopodia, which have identified roles in cellular motility and guidance, developing adhesions, and transmission of signals related to cellular motility [18]. By statistical analysis of variables contributing to shape in high resolution images, filopodia have been characterized and thus, identified and distinguished from other protrusion types [19]. Study of the neuronal growth cone revealed that filopodia are organized by an organelle called the focal actin ring, and have focal contacts at their basal, mid, and tip regions; the focal actin ring is believed to attach actin filaments to the basal region, and as a result, engender tension and filopodia emergence [20, 21]. Another type of actin-based cell protrusion observed in oncogenically transformed cells is the large protrusion. Like filopodia, it can be identified and distinguished from other types of protrusions via mathematical and statistical analysis of cell images [22]. Since large protrusion formation requires coordination over a comparably broader expanse of cytoplasm, the underlying mechanisms and effectors may be different from those in filopodia formation. It is through the dynamic assembly and disassembly of the protrusions' focal contacts that a cell is able to move. The PAK isoforms 1 (alpha), 2 (gamma), and 3 (beta) The PAK 1-3 isoforms were first identified in a search for binding partners for the GTPases Rac and Cdc42 [1]. Proteins known as p21s resemble the alpha subunit of heterotrimeric G proteins. To date, a total of six isoforms of PAK have been identified [23], with homologs of 3 PAK found in organisms ranging from protozoa to yeast to fruit flies to mammals [24, 25]. While sharing some similar features, PAK 1-3 isoforms can be distinguished from the PAK 4-6 isoforms: the former have an N-terminal inhibitory domain and are able to be activated by GTPases, while the latter lack a complex N-terminal sequence [26]. In the following, I will be focusing only on PAK 1-3 isoforms. PAK 1-3 are serine-threonine protein kinases [1]. In mammals, PAK 1 is highly expressed in the brain, muscles, and spleen. PAK 2 is expressed throughout the body. PAK 3 is highly expressed in the brain [1, 27]. PAK 1-3 have been found to have a role regulating the eukaryotic cell cytoskeleton [26]. Further characterization of their basic structure and modes of activation and inactivation will be presented in the following. As Figure 1 shows for PAK 1, PAK isoforms 1-3 all contain a N-terminal that is a regulatory domain and a C-terminal that is the catalytic domain. Differences in the number of PXXP SH3- binding motifs distinguish the three isoforms, with isoform 1 (alpha) having five, isoform 2 (gamma) having two, and isoform 3 (beta) having four, respectively [26]. All have one non- classical SH3 binding motif that interacts with PIX family proteins. PIX belongs to a class of proteins known as guanine nucleotide exchange factors, which activate a GTPase by exchanging guanine disphosphate (GDP) for guanine triphosphate (GTP) on the GTPase [16].