Alan Howe - Profile on Academia.edu (original) (raw)

Papers by Alan Howe

bioRxiv (Cold Spring Harbor Laboratory), Aug 19, 2023

Focal adhesions (FAs) form the junction between extracellular matrix (ECM)-bound integrins and th... more Focal adhesions (FAs) form the junction between extracellular matrix (ECM)-bound integrins and the actin cytoskeleton and also transmit signals that regulate cell adhesion, cytoskeletal dynamics, and cell migration. While many of these signals are rooted in reversible tyrosine phosphorylation, phosphorylation of FA proteins on Ser/Thr residues is far more abundant yet its mechanisms and consequences are far less understood. The cAMP-dependent protein kinase (protein kinase A; PKA) has important roles in cell adhesion and cell migration and is both an effector and regulator of integrin-mediated adhesion to the ECM. Importantly, subcellular localization plays a critically important role in specifying PKA function. Here, we show that PKA is present in isolated FA-cytoskeleton complexes and, using a targeted biosensor, active within FAs in live cells. Furthermore, using kinase-catalyzed biotinylation of isolated FA-cytoskeleton complexes, we identify fifty-three high-stringency candidate PKA substrates within FAs. From this list, we validate tensin-3 (Tns3) -a well-established molecular scaffold, regulator of cell migration, and component of focal and fibrillar adhesions -as a novel direct substrate for PKA. These observations identify a new pathway for phospho-regulation of Tns3 and, importantly, establish a new and important niche for localized PKA signaling and thus provide a foundation for further investigation of the role of PKA in the regulation of FA dynamics and signaling.

Translational cancer research, Feb 13, 2015

With increasing insights into the molecular landscape of ovarian cancer, subtypes are emerging th... more With increasing insights into the molecular landscape of ovarian cancer, subtypes are emerging that might require differential targeted therapies. While the combination of a platinum and a taxane remains the standard of care, newer therapies, specifically targeted to molecular anomalies, are rapidly being tested in various cancers. A major effort to better understand ovarian cancer occurred through the Cancer Atlas Project. The Catalogue of Somatic Mutations in Cancer (COSMIC) is a database that collates mutation data and associated information extracted from the primary literature. The information from the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), which systematically analyzed hundreds of ovarian cancer, are included in COSMIC, sometimes with discordant results. In this manuscript, the published data (mainly from TCGA) on somatic high grade papillary serous ovarian cancer (HGSOC) mutations has been used as the basis to propose a more granular approach to ovarian cancer treatment, already a reality for tumors such as lung and breast cancers. TP53 mutations are the most common molecular anomaly in HGSOC, and lead to genomic instability, perhaps through the FOXM1 node. Normalizing P53 has been a therapeutic challenge, and is being extensively studied. BRCAness is found an about 50% of HGSOC and can be targeted with poly (ADP-ribose) polymerase (PARP) inhibitors, such as olaparib, recently approved for ovarian cancer treatment. Other less common mutations have been found (MECOM, MAP15, CCNE1). Common mutations in other cancer (PI3K pathway, ERK kinase pathway, and EGFR family) are uncommon in ovarian cancer.

The cAMP-dependent protein kinase (Protein Kinase A; PKA) is a ubiquitous, promiscuous kinase who... more The cAMP-dependent protein kinase (Protein Kinase A; PKA) is a ubiquitous, promiscuous kinase whose activity is focused and specified through subcellular localization mediated by A-kinase anchoring proteins (AKAPs). PKA has complex roles as both an effector and a regulator of integrin-mediated cell adhesion to the extracellular matrix (ECM). Recent observations demonstrate that PKA is an active component of focal adhesions (FA), intracellular complexes coupling ECM-bound integrins to the actin cytoskeleton, suggesting the existence of one or more FA AKAPs. Using a combination of a promiscuous biotin ligase fused to PKA type-IIα regulatory (RIIα) subunits and subcellular fractionation, we identify the archetypal FA protein talin1 as an AKAP. Talin is a large, mechanosensitive scaffold that directly links integrins to actin filaments and promotes FA assembly by recruiting additional components in a force-dependent manner. The rod region of talin1 consists of 62 α-helices bundled into ...

ABSTRACTProtein Kinase A (PKA) is a pleiotropic serine/threonine kinase whose localized and dynam... more ABSTRACTProtein Kinase A (PKA) is a pleiotropic serine/threonine kinase whose localized and dynamic activity is required for cellular migration. Several cell types have shown robust PKA activity in the leading edge during migration. This activity is regulated by changes in actomyosin contractility, but the mechanism of the mechanochemical regulation of PKA remains unclear. Our ongoing investigation into this mechanism led us to discover a novel relationship between PKA and the non-receptor tyrosine kinase Focal Adhesion Kinase (FAK). Here, we find that while inhibition of actomyosin contractility leads to a relatively slow decrease in total cellular levels of phosphorylated FAK over tens of minutes, it decreases the focal adhesion-associated pool of both phospho-FAK and phospho-paxillin within two minutes, similar to the timing of the effect of inhibition of contractility on leading edge PKA activity. We then show that pharmacologic inhibition of FAK rapidly decreases leading edge P...

Frontiers in Molecular Biosciences

Cell migration requires establishment and maintenance of directional polarity, which in turn requ... more Cell migration requires establishment and maintenance of directional polarity, which in turn requires spatial heterogeneity in the regulation of protrusion, retraction, and adhesion. Thus, the signaling proteins that regulate these various structural processes must also be distinctly regulated in subcellular space. Protein Kinase A (PKA) is a ubiquitous serine/threonine kinase involved in innumerable cellular processes. In the context of cell migration, it has a paradoxical role in that global inhibition or activation of PKA inhibits migration. It follows, then, that the subcellular regulation of PKA is key to bringing its proper permissive and restrictive functions to the correct parts of the cell. Proper subcellular regulation of PKA controls not only when and where it is active but also specifies the targets for that activity, allowing the cell to use a single, promiscuous kinase to exert distinct functions within different subcellular niches to facilitate cell movement. In this ...

Translational cancer research, 2015

The focal adhesion protein talin is a mechanically gated A-kinase anchoring protein

Proceedings of the National Academy of Sciences of the United States of America, Mar 21, 2024

There is increasing interest in the ability of proteins involved in cell adhesion to convert mech... more There is increasing interest in the ability of proteins involved in cell adhesion to convert mechanical force into altered biochemistry. Cellular signal transduction is most often conducted through multiprotein scaffolds that consolidate, localize, and specify signaling inputs and outputs. This report bridges these fields by identifying an interaction between talin, a mechanosensitive adhesion protein, and PKA, a pleiotropic kinase with myriad cellular targets. Together, these observations form the foundation for a mechanotransduction pathway that utilizes forcedependent changes in protein conformation to establish a solid-state signaling complex well positioned to couple cellular tension to cellular communication.

Journal of Cellular Physiology, Oct 4, 2012

In areolar "loose" connective tissue, fibroblasts remodel their cytoskeleton within minutes in re... more In areolar "loose" connective tissue, fibroblasts remodel their cytoskeleton within minutes in response to static stretch resulting in increased cell body cross-sectional area that relaxes the tissue to a lower state of resting tension. It remains unknown whether the loosely arranged collagen matrix, characteristic of areolar connective tissue, is required for this cytoskeletal response to occur. The purpose of this study was to evaluate cytoskeletal remodeling of fibroblasts in and dissociated from areolar and dense connective tissue in response to 2 hours of static stretch in both native tissue and collagen gels of varying crosslinking. Rheometric testing indicated that the areolar connective tissue had a lower dynamic modulus and was more viscous than the dense connective tissue. In response to stretch, cells within the more compliant areolar connective tissue adopted a large "sheet-like" morphology that was in contrast to the smaller dendritic morphology in the dense connective tissue. By adjusting the in vitro collagen crosslinking, and the resulting dynamic modulus, it was demonstrated that cells dissociated from dense connective tissue are capable of responding when seeded into a compliant matrix, while cells dissociated from areolar connective tissue can lose their ability to respond when their matrix becomes stiffer. This set of experiments indicated stretch-induced fibroblast expansion was dependent on the distinct matrix material properties of areolar connective tissues as opposed to the cells' tissue of origin. These results also suggest that disease and pathological processes with increased crosslinks, such as diabetes and fibrosis, could impair fibroblast responsiveness in connective tissues.

Free Radical Biology and Medicine, Nov 1, 2014

Pharmacological ascorbate (AscH) induces selective cytotoxicity in pancreatic cancer cells vs. no... more Pharmacological ascorbate (AscH) induces selective cytotoxicity in pancreatic cancer cells vs. normal cells via the generation of extracellular hydrogen peroxide (H2O2). The resulting flux of H2O2, combined with a decreased antioxidant reserve in cancer cells, produces double-stranded DNA breaks and ultimately cell death. Redox active metal catalysts enhance ascorbate-induced cytotoxicity by increasing the rate of AscH oxidation and therefore the rate of H2O2 generated. It has previously been demonstrated that catalytic manganoporphyrins (MnPs) increase ascorbateinduced tumor cytotoxicity via this mechanism (Cancer Res. 73:1-10, 2013). We hypothesized that combining MnPs and AscH with the standard of care chemotherapeutic gemcitabine (Gem) would further enhance pancreatic cancer cell cytotoxicity without increasing normal tissue toxicity. In this study, MnPs were combined with AscH and administered with or without gemcitabine to three human pancreatic cancer cell lines, as well as immortalized normal pancreatic ductal epithelial cells. The MnPs MnT2EPyP (MnT2) and MnT4MPyP (MnT4) were investigated. Clonogenic survival was significantly decreased in pancreatic cancer cells treated with AscH + MnP + Gem, whereas normal cells were relatively resistant. Consistent with this result, the concentration of oxidized ascorbate radical (Asc) was significantly increased in treatment groups containing MnP and AscH. Furthermore, double stranded DNA breaks were increased in Gem-treated cells upon the addition of AscH + MnP. These findings were abrogated by extracellular catalase, further supporting the role of H2O2 flux in AscH + MnP-mediated cytotoxicity. Finally, tumor growth was significantly inhibited and survival increased in mice with pre-established pancreatic tumors treated with AscH , MnT2, and Gem compared to untreated controls, Gem therapy alone, or combination MnP + AscH therapies. Additionally, no evidence of increased systemic oxidative stress in the heart, liver or kidneys could be detected by 4-HNE-lipid peroxidation analysis, further supporting the specificity of treatment toward tumor cells. These results suggest a promising role for the use of MnPs in combination with pharmacologic AscH and standard of care chemotherapeutics in the treatment of pancreatic cancer.

Biochemical and Biophysical Research Communications, May 1, 2012

High matrix strains in the intervertebral disc occur during physiological motions and are amplifi... more High matrix strains in the intervertebral disc occur during physiological motions and are amplified around structural defects in the annulus fibrosus (AF). It remains unknown if large matrix strains in the human AF result in localized cell death. This study investigated strain amplitudes and substrate conditions where AF cells were vulnerable to stretch-induced apoptosis. Human degenerated AF cells were subjected to 1Hz-cyclic tensile strains for 24 hours on uniformly collagen coated substrates and on substrates with 40 μm stripes of collagen that restricted cellular reorientation. AF cells were capable of responding to stretch (stress fibers and focal adhesions aligned perpendicular to the direction of stretch), but were vulnerable to stretch-induced apoptosis when cytoskeletal reorientation was restricted, as could occur in degenerated states due to fibrosis and crosslink accumulation and at areas where high strains occur (around structural defects, delaminations, and herniations).

Molecular Biology of the Cell, Sep 1, 2016

Molecular Biology of the Cell MBoC | ARTICLE AMPK activity regulates trafficking of mitochondria ... more Molecular Biology of the Cell MBoC | ARTICLE AMPK activity regulates trafficking of mitochondria to the leading edge during cell migration and matrix invasion ABSTRACT Cell migration is a complex behavior involving many energy-expensive biochemical events that iteratively alter cell shape and location. Mitochondria, the principal producers of cellular ATP, are dynamic organelles that fuse, divide, and relocate to respond to cellular metabolic demands. Using ovarian cancer cells as a model, we show that mitochondria actively infiltrate leading edge lamellipodia, thereby increasing local mitochondrial mass and relative ATP concentration and supporting a localized reversal of the Warburg shift toward aerobic glycolysis. This correlates with increased pseudopodial activity of the AMP-activated protein kinase (AMPK), a critically important cellular energy sensor and metabolic regulator. Furthermore, localized pharmacological activation of AMPK increases leading edge mitochondrial flux, ATP content, and cytoskeletal dynamics, whereas optogenetic inhibition of AMPK halts mitochondrial trafficking during both migration and the invasion of three-dimensional extracellular matrix. These observations indicate that AMPK couples local energy demands to subcellular targeting of mitochondria during cell migration and invasion. INTRODUCTION Cell movement is a complex, highly dynamic process that integrates myriad diverse biochemical events to iteratively reshape and relocate the entire cell (Ridley et al., 2003). Execution of these events involves considerable expenditure of cellular energy, and recent studies demonstrate a critical role for energy-sensing pathways in various modes of migration (Nakano and Takashima, 2012). Within the protrusive leading edge alone, the consumption of ATP through sequestration and/or hydrolysis by actin monomers, molecular motors, and regulatory kinases could, if not replenished, generate a local ATP deficit that could halt cytoskeletal dynamics and cell migration. This paradigm can be further extended to invadopodia, lobopodia, and similar structures formed during migration and invasion in three dimensions, wherein the increased complexity of altering and penetrating through a three-dimensional (3D) extracellular matrix (ECM) requires increased coordination of multiple intracellular signaling networks with dynamic metabolic demands (Green and Yamada, 2007; Han et al., 2013). It is reasonable, then, to expect that such localized energy demands may be most efficiently met by a localized energy source rather than reliance on diffusion from central sources. Mitochondria are essential eukaryotic organelles responsible for, among other functions, generation of reactive oxygen species, regulation of cytosolic Ca 2+ transients, and production of the largest pool of cellular ATP (Nunnari and Suomalainen, 2012; Sena and Chandel, 2012). Mitochondria form a reticular network that radiates from the microtubule-organizing center near the nucleus to supply central and distal regions of the cell with essential energy and metabolites. The morphology of this network is dynamically regulated

Abstract 5224: Non-canonical activity of threonyl-tRNA synthetase promotes angiogenesis and invasion in a mouse model of ovarian cancer

Despite clear connections between the tumor microenvironment and ovarian cancer invasion, the und... more Despite clear connections between the tumor microenvironment and ovarian cancer invasion, the underlying molecular mechanisms remain elusive. We have recently shown that the protein synthesis regulator threonyl-tRNA synthetase (TARS) has a unique extracellular angiogenic activity separate from its canonical function. Secreted TARS promotes endothelial cell migration and tube formation, and a selective TARS inhibitor, BC194, disrupts normal vessel development in zebra fish and chick embryo models. TARS expression also correlates with tumor stage and angiogenesis in human ovarian cancer. The objective of this study was to validate the biological and clinical relationship between TARS and ovarian tumor progression using a syngenic mouse model of epithelial ovarian cancer. Tumors were initiated by intraperitoneal injection of ID8 mouse ovarian cancer cells, and tumors formed at 4-6 weeks were scored for invasiveness and analyzed by immunostaining for TARS expression and microvascular density. To test the hypothesis that overexpression of TARS promotes invasion, cells were stably transfected with a TARS expression plasmid prior to injection. To test the hypothesis that TARS inhibition reduces tumor invasion, animals harboring ID8 tumors were treated with the high-affinity TARS inhibitor BC194. We found that TARS levels were elevated in ovarian tumors as compared with normal ovarian tissue. Overexpression of TARS in ID8 cells also resulted in enhanced invasiveness and microvascular density of the resulting tumors (p = 0.026). Preliminary results also indicated that inhibition of TARS by BC194 treatment reduced tumor angiogenesis and growth (p = 0.005) without observed toxicities in the animals. Overall, these results show that modifying TARS expression or activity can affect in vivo ovarian tumor angiogenesis and progression. These results encourage further study of TARS as a regulator of the tumor microenvironment and as a possible target for diagnosis and treatment of ovarian cancer. Citation Format: Peibin Wo, Theresa Wellman, Alan Howe, Christopher Francklyn, Karen M. Lounsbury. Non-canonical activity of threonyl-tRNA synthetase promotes angiogenesis and invasion in a mouse model of ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5224. doi:10.1158/1538-7445.AM2015-5224

Carolina Digital Repository (University of North Carolina at Chapel Hill), 2003

ntegrins coordinate spatial signaling events essential for cell polarity and directed migration. ... more ntegrins coordinate spatial signaling events essential for cell polarity and directed migration. Such signals from ␣ 4 integrins regulate cell migration in development and in leukocyte trafficking. Here, we report that efficient ␣ 4mediated migration requires spatial control of ␣ 4 phosphorylation by protein kinase A, and hence localized inhibition of binding of the signaling adaptor, paxillin, to the integrin. In migrating cells, phosphorylated ␣ 4 accumulated along the leading edge. Blocking ␣ 4 phosphorylation by mutagenesis or by inhibition of protein kinase A drastically reduced ␣ 4-Î dependent migration and lamellipodial stability. ␣ 4 phosphorylation blocks paxillin binding in vitro; we now find that paxillin and phospho-␣ 4 were in distinct clusters at the leading edge of migrating cells, whereas unphosphorylated ␣ 4 and paxillin colocalized along the lateral edges of those cells. Furthermore, enforced paxillin association with ␣ 4 inhibits migration and reduced lamellipodial stability. These results show that topographically specific integrin phosphorylation can control cell migration and polarization by spatial segregation of adaptor protein binding.

Scientific Reports, 2018

There is growing appreciation of the importance of the mechanical properties of the tumor microen... more There is growing appreciation of the importance of the mechanical properties of the tumor microenvironment on disease progression. However, the role of extracellular matrix (ECM) stiffness and cellular mechanotransduction in epithelial ovarian cancer (EOC) is largely unknown. Here, we investigated the effect of substrate rigidity on various aspects of SKOV3 human EOC cell morphology and migration. Young’s modulus values of normal mouse peritoneum, a principal target tissue for EOC metastasis, were determined by atomic force microscopy (AFM) and hydrogels were fabricated to mimic these values. We find that cell spreading, focal adhesion formation, myosin light chain phosphorylation, and cellular traction forces all increase on stiffer matrices. Substrate rigidity also positively regulates random cell migration and, importantly, directional increases in matrix tension promote SKOV3 cell durotaxis. Matrix rigidity also promotes nuclear translocation of YAP1, an oncogenic transcription ...

Free Radical Biology and Medicine, 2014

Journal of Cellular Physiology, 2011

The visco‐elastic behavior of connective tissue is generally attributed to the material propertie... more The visco‐elastic behavior of connective tissue is generally attributed to the material properties of the extracellular matrix rather than cellular activity. We have previously shown that fibroblasts within areolar connective tissue exhibit dynamic cytoskeletal remodeling within minutes in response to tissue stretch ex vivo and in vivo. Here, we tested the hypothesis that fibroblasts, through this cytoskeletal remodeling, actively contribute to the visco‐elastic behavior of the whole tissue. We measured significantly increased tissue tension when cellular function was broadly inhibited by sodium azide and when cytoskeletal dynamics were compromised by disrupting microtubules (with colchicine) or actomyosin contractility (via Rho kinase inhibition). These treatments led to a decrease in cell body cross‐sectional area and cell field perimeter (obtained by joining the end of all of a fibroblast's processes). Suppressing lamellipodia formation by inhibiting Rac‐1 decreased cell body...

Journal of Biological Chemistry, 1998

S1687 Serotonin Transporter Function and Expression Are Up-Regulated By a PKA-Independent cAMP Signaling Pathway in Intestinal Epithelial Cells

Gastroenterology, Apr 1, 2008

Dynamic subcellular regulation of Protein kinase A (PKA) activity is important for the motile beh... more Dynamic subcellular regulation of Protein kinase A (PKA) activity is important for the motile behavior of many cell types, yet the mechanisms governing PKA activity during cell migration remain largely unknown. The motility of SKOV-3 epithelial ovarian cancer (EOC) cells has been shown to be dependent on both localized PKA activity and, more recently, on mechanical reciprocity between cellular tension and extracellular matrix (ECM) rigidity. Here, we investigated the possibility that PKA is regulated by mechanical signaling during migration. We find that localized PKA activity in migrating cells rapidly decreases upon inhibition of actomyosin contractility (specifically, of myosin ATPase, ROCK (Rho kinase), or MLCK (myosin light chain kinase) activity). Moreover, PKA activity is spatially and temporally correlated with cellular traction forces in migrating cells. Additionally, PKA is rapidly and locally activated by mechanical stretch in an actomyosin contractility-dependent manner....

Scientific reports, Jan 14, 2015

Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate... more Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the...