PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (1021131)

Clipboard (0)
None

Related Articles

1.  Paxillin-dependent Paxillin Kinase Linker and p21-Activated Kinase Localization to Focal Adhesions Involves a Multistep Activation Pathway 
Molecular Biology of the Cell  2002;13(5):1550-1565.
The precise temporal-spatial regulation of the p21-activated serine-threonine kinase PAK at the plasma membrane is required for proper cytoskeletal reorganization and cell motility. However, the mechanism by which PAK localizes to focal adhesions has not yet been elucidated. Indirect binding of PAK to the focal adhesion protein paxillin via the Arf-GAP protein paxillin kinase linker (PKL) and PIX/Cool suggested a mechanism. In this report, we demonstrate an essential role for a paxillin–PKL interaction in the recruitment of activated PAK to focal adhesions. Similar to PAK, expression of activated Cdc42 and Rac1, but not RhoA, stimulated the translocation of PKL from a generally diffuse localization to focal adhesions. Expression of the PAK regulatory domain (PAK1–329) or the autoinhibitory domain (AID 83–149) induced PKL, PIX, and PAK localization to focal adhesions, indicating a role for PAK scaffold activation. We show PIX, but not NCK, binding to PAK is necessary for efficient focal adhesion localization of PAK and PKL, consistent with a PAK–PIX–PKL linkage. Although PAK activation is required, it is not sufficient for localization. The PKL amino terminus, containing the PIX-binding site, but lacking paxillin-binding subdomain 2 (PBS2), was unable to localize to focal adhesions and also abrogated PAK localization. An identical result was obtained after PKLΔPBS2 expression. Finally, neither PAK nor PKL was capable of localizing to focal adhesions in cells overexpressing paxillinΔLD4, confirming a requirement for this motif in recruitment of the PAK–PIX–PKL complex to focal adhesions. These results suggest a GTP-Cdc42/GTP-Rac triggered multistep activation cascade leading to the stimulation of the adaptor function of PAK, which through interaction with PIX provokes a functional PKL PBS2–paxillin LD4 association and consequent recruitment to focal adhesions. This mechanism is probably critical for the correct subcellular positioning of PAK, thereby influencing the ability of PAK to coordinate cytoskeletal reorganization associated with changes in cell shape and motility.
doi:10.1091/mbc.02-02-0015
PMCID: PMC111126  PMID: 12006652
2.  Group I p21-activated kinases regulate thyroid cancer cell migration and are overexpressed and activated in thyroid cancer invasion 
Endocrine-related cancer  2010;17(4):989-999.
p21-activated kinases (PAKs) are a family of serine/threonine kinases that regulate cytoskeletal dynamics and cell motility. PAKs are subdivided into group I (PAKs 1–3) and group II (PAKs 4–6) on the basis of structural and functional characteristics. Based on prior gene expression data that predicted enhanced PAK signaling in the invasive fronts of aggressive papillary thyroid cancers (PTCs), we hypothesized that PAKs functionally regulate thyroid cancer cell motility and are activated in PTC invasive fronts. We examined PAK isoform expression in six human thyroid cancer cell lines (BCPAP, KTC1, TPC1, FTC133, C643, and SW1746) by quantitative reverse transcription-PCR and western blot. All cell lines expressed PAKs 1–4 and PAK6 mRNA and PAKs 1–4 protein; PAK6 protein was variably expressed. Samples from normal and malignant thyroid tissues also expressed PAKs 1–4 and PAK6 mRNA; transfection with the group I (PAKs 1–3) PAK-specific p21 inhibitory domain molecular inhibitor reduced transwell filter migration by ~50% without altering viability in all cell lines (P<0.05). BCPAP and FTC133 cells were transfected with PAK1, PAK2, or PAK3-specific small interfering RNA (siRNA); only PAK1 siRNA reduced migration significantly for both cell lines. Immunohistochemical analysis of seven invasive PTCs demonstrated an increase in PAK1 and pPAK immunoactivity in the invasive fronts versus the tumor center. In conclusion, PAK isoforms are expressed in human thyroid tissues and cell lines. PAK1 regulates thyroid cancer cell motility, and PAK1 and pPAK levels are increased in PTC invasive fronts. These data implicate PAKs as regulators of thyroid cancer invasion.
doi:10.1677/ERC-10-0168
PMCID: PMC3717591  PMID: 20817787
3.  Activation of P21-activated protein kinase 2 is an independent prognostic predictor for patients with gastric cancer 
Diagnostic Pathology  2014;9:55.
Objective
p21-activated kinase (PAK) 2, as a member of the PAK family kinases, is involved in a number of hallmark processes including cell proliferation, survival, mitosis, apoptosis, motility and angiogenesis. However, the clinical significance of the activation of PAK2 in human gastric cancer has not been fully elucidated. The aim of this study was to investigate whether PAK2 expression and its phosphorylation status are correlated with tumor progression and prognosis in gastric cancer.
Methods
Expression patterns and subcellular localizations of PAK2 and Ser20-phosphorylated PAK2 (pSer20PAK2) in 82 gastric cancer patients were detected by immunohistochemistry.
Results
Both PAK2 and pSer20PAK2 immunostainings were localized in the cytoplasm of tumor cells of gastric cancer tissues. Compared with the normal gastric mucosa, the expression levels of PAK2 and pSer20PAK2 proteins were both significantly increased (both P < 0.001). Additionally, the patients displaying the over-expression of PAK2 and pSer20PAK2 proteins were dramatically associated with unfavorable clinicopathologic variables including higher tumor depth (P = 0.022 and 0.036, respectively), greater extent of lymph node metastasis ((P = 0.022 and 0.036, respectively), positive distant metastasis (P = 0.025 and 0.038, respectively) and advanced tumor stage (P = 0.018 and 0.031, respectively). Moreover, the patients overexpressing PAK2 and pSer20PAK2 proteins have poor overall survival rates relative to those without overexpression of these proteins. Furthermore, cox multi-factor analysis showed that PAK2 (p = 0.012) and pSer20PAK2 (p = 0.010) were independent prognosis factors for human gastric cancer.
Conclusion
Our data suggest for the first time that PAK2 activation may be associated with advanced tumor progression and poor prognosis of gastric cancer.
Virtual slides
The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1236344107120406.
doi:10.1186/1746-1596-9-55
PMCID: PMC3975179  PMID: 24621074
Gastric cancer; p21-activated kinase 2; Phosphorylation; Immunohistochemistry; Prognosis
4.  P21-Activated Kinase 4 Overexpression in Metastatic Gastric Cancer Patients 
Translational Oncology  2011;4(6):345-349.
INTRODUCTION: P21-activated kinase 4 (PAK), a subfamily of serine/threonine kinases originally known as a regulator of cytoskeletal dynamics and cell motility, has recently been revealed to play a key role in oncogenic signaling pathways. We studied the frequency and clinical features of PAK4-overexpressed metastatic gastric cancer. PATIENTS AND METHODS: PAK4 overexpression was screened by Western blot in 18 human gastric cancer cell lines. Immunohistochemical staining of PAK4 protein was performed in tumor specimens of 49 metastatic gastric cancer patients who received palliative capecitabine/cisplatin as first-line treatment. RESULTS: PAK4 protein overexpression was detected strongly in five gastric cell lines (AGS, MGK-28, MKN-74, SNU-216, SNU-601) and weakly in four cell lines (KATOIII, MKN-1, SNU-620, and SNU-719). PAK4 knockdown by small interfering RNA induced apoptosis in PAK4-overexpressed AGS gastric cancer cells. Immunohistochemical staining revealed PAK4 overexpressions in 4 (8.1%) of 49 metastatic gastric cancer specimens. None of the four patients with PAK4(+) responded to capecitabine/cisplatin chemotherapy, and PAK4(+) gastric cancer patients had a trend of poorer survival compared with PAK(-)(P = .876). CONCLUSIONS: We demonstrated PAK4 overexpression in a subset of gastric cancer patients, implicating a role in gastric cancer tumorigenesis. Its prognostic significance and efficacy as a drug target should be further studied.
PMCID: PMC3243657  PMID: 22190998
5.  Nucleocytoplasmic Shuttling of Pak5 Regulates Its Antiapoptotic Properties†  
Molecular and Cellular Biology  2006;26(8):3215-3230.
p21-activated kinase 5 (Pak5) is an effector for the small GTPase Cdc42, known to activate cell survival signaling pathways. Previously, we have shown that Pak5 localizes primarily to mitochondria. To study the relationship between Pak5 localization and its effects on apoptosis, we identified three N-terminal regions that regulate the localization of this kinase: a mitochondrial targeting sequence, a nuclear export sequence, and a nuclear localization sequence. When the first two sequences are deleted, Pak5 is retained in the nucleus and no longer protects cells from apoptosis. Moreover, blockade of nuclear export with leptomycin B causes endogenous Pak5 to accumulate in the nucleus. Additionally, the removal of the N-terminal nuclear localization sequence abolishes Pak5 translocation to the nucleus. Finally, we show that reduction of endogenous Pak5 expression in neuroblastoma and neural stem cells increases their sensitivity to apoptosis and that this effect is reversed upon reexpression of wild-type Pak5 but not of a mutant form of Pak5 that cannot localize to mitochondria. These results show that Pak5 shuttles from mitochondria to the nucleus and that the mitochondrial localization of Pak5 is vital to its effects on cell survival.
doi:10.1128/MCB.26.8.3215-3230.2006
PMCID: PMC1446938  PMID: 16581795
6.  Integrated analysis of breast cancer cell lines reveals unique signaling pathways 
Genome Biology  2009;10(3):R31.
Mapping of sub-networks in the EGFR-MAPK pathway in different breast cancer cell lines reveals that PAK1 may be a marker for sensitivity to MEK inhibitors.
Background
Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EgfR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes.
Results
We were interested in identifying subnetworks within the EgfR-MAPK pathway that are similarly deregulated across subsets of breast cancers. To that end, we mapped genomic, transcriptional and proteomic profiles for 30 breast cancer cell lines onto a curated Pathway Logic symbolic systems model of EgfR-MAPK signaling. This model was composed of 539 molecular states and 396 rules governing signaling between active states. We analyzed these models and identified several subtype-specific subnetworks, including one that suggested Pak1 is particularly important in regulating the MAPK cascade when it is over-expressed. We hypothesized that Pak1 over-expressing cell lines would have increased sensitivity to Mek inhibitors. We tested this experimentally by measuring quantitative responses of 20 breast cancer cell lines to three Mek inhibitors. We found that Pak1 over-expressing luminal breast cancer cell lines are significantly more sensitive to Mek inhibition compared to those that express Pak1 at low levels. This indicates that Pak1 over-expression may be a useful clinical marker to identify patient populations that may be sensitive to Mek inhibitors.
Conclusions
All together, our results support the utility of symbolic system biology models for identification of therapeutic approaches that will be effective against breast cancer subsets.
doi:10.1186/gb-2009-10-3-r31
PMCID: PMC2691002  PMID: 19317917
7.  The p21-Activated Kinase (PAK) Family Member PakD Is Required for Chemorepulsion and Proliferation Inhibition by Autocrine Signals in Dictyostelium discoideum 
PLoS ONE  2014;9(5):e96633.
In Dictyostelium discoideum, the secreted proteins AprA and CfaD function as reporters of cell density and regulate cell number by inhibiting proliferation at high cell densities. AprA also functions to disperse groups of cells at high density by acting as a chemorepellent. However, the signal transduction pathways associated with AprA and CfaD are not clear, and little is known about how AprA affects the cytoskeleton to regulate cell movement. We found that the p21-activated kinase (PAK) family member PakD is required for both the proliferation-inhibiting activity of AprA and CfaD and the chemorepellent activity of AprA. Similar to cells lacking AprA or CfaD, cells lacking PakD proliferate to a higher cell density than wild-type cells. Recombinant AprA and CfaD inhibit the proliferation of wild-type cells but not cells lacking PakD. Like AprA and CfaD, PakD affects proliferation but does not significantly affect growth (the accumulation of mass) on a per-nucleus basis. In contrast to wild-type cells, cells lacking PakD are not repelled from a source of AprA, and colonies of cells lacking PakD expand at a slower rate than wild-type cells, indicating that PakD is required for AprA-mediated chemorepulsion. A PakD-GFP fusion protein localizes to an intracellular punctum that is not the nucleus or centrosome, and PakD-GFP is also occasionally observed at the rear cortex of moving cells. Vegetative cells lacking PakD show excessive actin-based filopodia-like structures, suggesting that PakD affects actin dynamics, consistent with previously characterized roles of PAK proteins in actin regulation. Together, our results implicate PakD in AprA/CfaD signaling and show that a PAK protein is required for proper chemorepulsive cell movement in Dictyostelium.
doi:10.1371/journal.pone.0096633
PMCID: PMC4010531  PMID: 24797076
8.  Regulation of Macropinocytosis by p21-activated Kinase-1 
Molecular Biology of the Cell  2000;11(10):3341-3352.
The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5′-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.
PMCID: PMC14996  PMID: 11029040
9.  PAK Signaling in Oncogenesis 
Oncogene  2009;28(28):2545-2555.
The p21-activated kinase (PAK) family of serine/threonine kinases plays a pivotal role in physiological processes including motility, survival, mitosis, transcription and translation. PAKs are evolutionally conserved and widely expressed in a variety of tissues and are often over expressed in multiple cancer types. Depending on structural and functional similarities, the six members of PAK family are divided into two groups with three members in each group. Group I PAKs are activated by extracellular signals through GTPase-dependent and independent mechanisms. In contrast, group II PAKs are constitutively active. Over the years, accumulating data from tissue culture models and human tumors has increased our understanding about the biology of PAK family members. In this review, we have summarized the complex regulation of PAK and its downstream diverse myriads of effectors which in-turn are responsible for the biologic effects of PAK family of kinases in cancer cells.
doi:10.1038/onc.2009.119
PMCID: PMC2731678  PMID: 19465939
PAK; cancer; cytoskeleton; survival; mitosis; knockout mice
10.  Interaction with LC8 Is Required for Pak1 Nuclear Import and Is Indispensable for Zebrafish Development 
PLoS ONE  2009;4(6):e6025.
Pak1 (p21 activated kinase 1) is a serine/threonine kinase implicated in regulation of cell motility and survival and in malignant transformation of mammary epithelial cells. In addition, the dynein light chain, LC8, has been described to cooperate with Pak1 in malignant transformation of breast cancer cells. Pak1 itself may aid breast cancer development by phosphorylating nuclear proteins, including estrogen receptor alpha. Recently, we showed that the LC8 binding site on Pak1 is adjacent to the nuclear localization sequence (NLS) required for Pak1 nuclear import. Here, we demonstrate that the LC8-Pak1 interaction is necessary for epidermal growth factor (EGF)-induced nuclear import of Pak1 in MCF-7 cells, and that this event is contingent upon LC8-mediated Pak1 dimerization. In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells. Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site. Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.
doi:10.1371/journal.pone.0006025
PMCID: PMC2698211  PMID: 19557173
11.  The PAKs come of age 
Cellular Logistics  2012;2(2):54-58.
Protein kinases are versatile signaling molecules that are involved in the regulation most physiological responses. The p21-activated kinases (PAKs) can be activated directly by the small GTPases Rac and Cdc42 and are among the best characterized downstream effectors of these Rho proteins. The structure, substrate specificity and functional role of PAKS are evolutionarily conserved from protozoa to mammals. Vertebrate PAKs are particularly important for cytoskeletal remodeling and focal adhesion assembly, thereby contributing to dynamic processes such as cell migration and synaptic plasticity. This issue of Cellular Logistics focuses on the PAK family of kinases, with ten reviews written by researchers currently working in the field. Here in this introductory overview we highlight some of the most interesting recent discoveries regarding PAK biochemistry and biology. The reviews in this issue cover a range of topics including the atomic structures of PAK1 and PAK4, their role in animals as assessed by knockout studies, and how PAKs are likely to contribute to cancer and neurodegenerative diseases. The promise remains that PAK inhibitors will emerge that validate current pre-clinical studies suggesting that blocking PAK activity will positively contribute to human health.
doi:10.4161/cl.22084
PMCID: PMC3485743  PMID: 23125949
12.  Potential Compensation among Group I PAK Members in Hindlimb Ischemia and Wound Healing 
PLoS ONE  2014;9(11):e112239.
PAKs are serine/threonine kinases that regulate cytoskeletal dynamics and cell migration. PAK1 is activated by binding to the small EF hand protein, CIB1, or to the Rho GTPases Rac1 or Cdc42. The role of PAK1 in angiogenesis was established based only on in vitro studies and its role in angiogenesis in vivo has never been examined. Here we tested the hypothesis that PAK1 is an essential regulator of ischemic neovascularization (arteriogenesis and angiogenesis) and wound healing using a global PAK1 knockout mouse. Neovascularization was assessed using unilateral hindlimb ischemia. We found that plantar perfusion, limb use and appearance were not significantly different between 6–8 week old PAK1−/− and PAK1+/+ mice throughout the 21-day period following hindlimb ischemia; however a slightly delayed healing was observed in 16 week old PAK1−/− mice. In addition, the wound healing rate, as assessed with an ear punch assay, was unchanged in PAK1−/− mice. Surprisingly, however, we observed a notable increase in PAK2 expression and phosphorylation in ischemic gastrocnemius tissue from PAK1−/− but not PAK1+/+ mice. Furthermore, we observed higher levels of activated ERK2, but not AKT, in ischemic and non-ischemic muscle of PAK1−/− mice upon hindlimb ischemic injury. A group I PAK inhibitor, IPA3, significantly inhibited endothelial cell sprouting from aortic rings in both PAK1−/− and PAK1+/+ mice, implying that PAK2 is a potential contributor to this process. Taken together, our data indicate that while PAK1 has the potential to contribute to neovascularization and wound healing, PAK2 may functionally compensate when PAK1 is deficient.
doi:10.1371/journal.pone.0112239
PMCID: PMC4224450  PMID: 25379771
13.  Constitutive p21-activated Kinase (PAK) Activation in Breast Cancer Cells as a Result of Mislocalization of PAK to Focal AdhesionsD⃞ 
Molecular Biology of the Cell  2004;15(6):2965-2977.
Cytoskeletal remodeling is critical for cell adhesion, spreading, and motility. p21-activated kinase (PAK), an effector molecule of the Rho GTPases Rac and Cdc42, has been implicated in cytoskeletal remodeling and cell motility. PAK kinase activity and subcellular distribution are tightly regulated by rapid and transient localized Rac and Cdc42 activation, and by interactions mediated by adapter proteins. Here, we show that endogenous PAK is constitutively activated in certain breast cancer cell lines and that this active PAK is mislocalized to atypical focal adhesions in the absence of high levels of activated Rho GTPases. PAK localization to focal adhesions in these cells is independent of PAK kinase activity, NCK binding, or GTPase binding, but requires the association of PAK with PIX. Disruption of the PAK–PIX interaction with competitive peptides displaces PAK from focal adhesions and results in a substantial reduction in PAK hyperactivity. Moreover, disruption of the PAK–PIX interaction is associated with a dramatic decrease of PIX and paxillin in focal adhesions, indicating that PAK localization to these structures via PIX is required for the maintenance of paxillin- and PIX-containing focal adhesions. Abnormal regulation of PAK localization and activity may contribute to the tumorigenic properties of certain breast cancer cells.
doi:10.1091/mbc.E03-08-0604
PMCID: PMC420118  PMID: 15047871
14.  Localization of p21-Activated Kinase 1 (PAK1) to Pinocytic Vesicles and Cortical Actin Structures in Stimulated Cells  
The Journal of Cell Biology  1997;138(6):1265-1278.
The mechanisms through which the small GTPases Rac1 and Cdc42 regulate the formation of membrane ruffles, lamellipodia, and filopodia are currently unknown. The p21-activated kinases (PAKs) are direct targets of active Rac and Cdc42 which can induce the assembly of polarized cytoskeletal structures when expressed in fibroblasts, suggesting that they may play a role in mediating the effects of these GTPases on cytoskeletal dynamics.
We have examined the subcellular localization of endogenous PAK1 in fibroblast cell lines using specific PAK1 antibodies. PAK1 is detected in submembranous vesicles in both unstimulated and stimulated fibroblasts that colocalize with a marker for fluid-phase uptake. In cells stimulated with PDGF, in v-Src–transformed fibroblasts, and in wounded cells, PAK1 redistributed into dorsal and membrane ruffles and into the edges of lamellipodia, where it colocalizes with polymerized actin. PAK1 was also colocalized with F-actin in membrane ruffles extended as a response to constitutive activation of Rac1. PAK1 appears to precede F-actin in translocating to cytoskeletal structures formed at the cell periphery. The association of PAK1 with the actin cytoskeleton is prevented by the actin filament-disrupting agent cytochalasin D and by the phosphatidylinositol 3-kinase inhibitor wortmannin. Co-immunoprecipitation experiments demonstrate an in vivo interaction of PAK1 with filamentous (F)-actin in stimulated cells. Microinjection of a constitutively active PAK1 mutant into Rat-1 fibroblasts overexpressing the insulin receptor (HIRcB cells) induced the formation of F-actin- and PAK1-containing structures reminiscent of dorsal ruffles. These data indicate a close correlation between the subcellular distribution of endogenous PAK1 and the formation of Rac/Cdc42-dependent cytoskeletal structures and support an active role for PAK1 in regulating cortical actin rearrangements.
PMCID: PMC2132543  PMID: 9298982
15.  p21-Activated Kinase 5 (Pak5) Localizes to Mitochondria and Inhibits Apoptosis by Phosphorylating BAD 
Molecular and Cellular Biology  2003;23(16):5526-5539.
Pak5 is the most recently identified and least understood member of the p21-activated kinase (Pak) family. This kinase is known to promote neurite outgrowth in vitro, but its localization, substrates, and effects on cell survival have not been reported. We show here that Pak5 has unique properties that distinguish it from all other members of the Pak family. First, Pak5, unlike Pak1, cannot complement an STE20 mutation in Saccharomyces cerevisiae. Second, Pak5 binds to the GTPases Cdc42 and Rac, but these GTPases do not regulate Pak5 kinase activity, which is constitutive and stronger than any other Pak. Third, Pak5 prevents apoptosis induced by camptothecin and C2-ceramide by phosphorylating BAD on Ser-112 in a protein kinase A-independent manner and prevents the localization of BAD to mitochondria, thereby inhibiting the apoptotic cascade that leads to apoptosis. Finally, we show that Pak5 itself is constitutively localized to mitochondria, and that this localization is independent of kinase activity or Cdc42 binding. These features make Pak5 unique among the Pak family and suggest that it plays an important role in apoptosis through BAD phosphorylation.
doi:10.1128/MCB.23.16.5526-5539.2003
PMCID: PMC166342  PMID: 12897128
16.  PAK signaling in cancer 
Cellular Logistics  2012;2(2):105-116.
Transformation of a normal cell to a cancer cell is caused by mutations in genes that regulate proliferation, apoptosis, and invasion. Small GTPases such as Ras, Rho, Rac and Cdc42 orchestrate many of the signals that are required for malignant transformation. The p21-activated kinases (PAKs) are effectors of Rac and Cdc42. PAKs are a family of serine/threonine protein kinases comprised of six isoforms (PAK1–6), and they play important roles in cytoskeletal dynamics, cell survival and proliferation. They act as key signal transducers in several cancer signaling pathways, including Ras, Raf, NFκB, Akt, Bad and p53. Although PAKs are not mutated in cancers, they are overexpressed, hyperactivated or amplified in several human tumors and their role in cell transformation make them attractive therapeutic targets. This review discusses the evidence that PAK is important for cell transformation and some key signaling pathways it regulates. This review primarily discusses Group I PAKs (PAK1, PAK2 and PAK3) as Group II PAKs (PAK4, PAK5 and PAK6) are discussed elsewhere in this issue (by Minden).
doi:10.4161/cl.21882
PMCID: PMC3490961  PMID: 23162742
cancer; amplification; PAK; p21 activated kinase; Rac; CDC42; protein kinase
17.  Molecular Pathways: Targeting P21-activated Kinase 1 Signaling in Cancer: Opportunities, Challenges and Limitations 
Clinical Cancer Research  2012;18(14):3743-3749.
The evolution of cancer cells involves deregulation of highly regulated fundamental pathways that are central to normal cellular architecture and functions. The p21-activated kinase 1 (PAK1) was initially identified as a downstream effector of the GTPases Rac and Cdc42. Subsequent studies uncovered a plethora of its new functions in growth factor and steroid receptor signaling, cytoskeleton remodeling, cell survival, oncogenic transformation, and gene transcription, largely through systematic discovery of its direct, physiologically relevant substrates. PAK1 is widely up-regulated in several human cancers including hormone-dependent cancer, and is intimately linked to tumor progression and therapeutic resistance. These exciting developments combined with the kinase-independent role of PAK1-centered phenotypic signaling in cancer cells elevated PAK1 as an attractive drug target. The structural and biochemical studies revealed the precise mechanism of PAK1 activation, offering the possibilities to develop PAK1-targeted cancer therapeutic approaches. In addition, emerging reports suggest the potential of PAK1 and its specific phosphorylated substrates as cancer prognostic markers. Here, we summarize the PAK1 molecular pathways in human cancer and discuss the current status of PAK1 targeted anti-cancer therapies.
doi:10.1158/1078-0432.CCR-11-1952
PMCID: PMC3399091  PMID: 22595609
PAK1; Growth factor signaling; Cytoskeleton; Oncogenic transformation; Chromatin and nuclear signaling; Protein kinase inhibitor
18.  Integrin-mediated Cell Attachment Induces a PAK4-dependent Feedback Loop Regulating Cell Adhesion through Modified Integrin αvβ5 Clustering and Turnover 
Molecular Biology of the Cell  2010;21(19):3317-3329.
This article presents a novel mechanism deployed by cells to tune cell adhesion levels through the autoinhibitory regulation of integrin adhesion involving the activation of PAK4.
Cell-to-extracellular matrix adhesion is regulated by a multitude of pathways initiated distally to the core cell–matrix adhesion machinery, such as via growth factor signaling. In contrast to these extrinsically sourced pathways, we now identify a regulatory pathway that is intrinsic to the core adhesion machinery, providing an internal regulatory feedback loop to fine tune adhesion levels. This autoinhibitory negative feedback loop is initiated by cell adhesion to vitronectin, leading to PAK4 activation, which in turn limits total cell–vitronectin adhesion strength. Specifically, we show that PAK4 is activated by cell attachment to vitronectin as mediated by PAK4 binding partner integrin αvβ5, and that active PAK4 induces accelerated integrin αvβ5 turnover within adhesion complexes. Accelerated integrin turnover is associated with additional PAK4-mediated effects, including inhibited integrin αvβ5 clustering, reduced integrin to F-actin connectivity and perturbed adhesion complex maturation. These specific outcomes are ultimately associated with reduced cell adhesion strength and increased cell motility. We thus demonstrate a novel mechanism deployed by cells to tune cell adhesion levels through the autoinhibitory regulation of integrin adhesion.
doi:10.1091/mbc.E10-03-0245
PMCID: PMC2947468  PMID: 20719960
19.  Identification of MYO18A as a Novel Interacting Partner of the PAK2/βPIX/GIT1 Complex and Its Potential Function in Modulating Epithelial Cell Migration 
Molecular Biology of the Cell  2010;21(2):287-301.
MYO18A is found as a novel PAK2 binding partner via βPIX/GIT1. MYO18A-depleted cells showed dramatic changes in shape, actin stress fiber and membrane ruffle formation, and displayed increases in the number and size of focal adhesions and a decrease in cell migration, suggesting an important role of MYO18A in regulating epithelial cell migration.
The p21-activated kinase (PAK) 2 is known to be involved in numerous biological functions, including the regulation of actin reorganization and cell motility. To better understand the mechanisms underlying this regulation, we herein used a proteomic approach to identify PAK2-interacting proteins in human epidermoid carcinoma A431 cells. We found that MYO18A, an emerging member of the myosin superfamily, is a novel PAK2 binding partner. Using a siRNA knockdown strategy and in vitro binding assay, we discovered that MYO18A binds to PAK2 through the βPIX/GIT1 complex. Under normal conditions, MYO18A and PAK2 colocalized in lamellipodia and membrane ruffles. Interestingly, knockdown of MYO18A in cells did not prevent formation of the PAK2/βPIX/GIT1 complex, but rather apparently changed its localization to focal adhesions. Moreover, MYO18A-depleted cells showed dramatic changes in morphology and actin stress fiber and membrane ruffle formation and displayed increases in the number and size of focal adhesions. Migration assays revealed that MYO18A-depleted cells had decreased cell motility, and reexpression of MYO18A restored their migration ability. Collectively, our findings indicate that MYO18A is a novel binding partner of the PAK2/βPIX/GIT1 complex and suggest that MYO18A may play an important role in regulating epithelial cell migration via affecting multiple cell machineries.
doi:10.1091/mbc.E09-03-0232
PMCID: PMC2808764  PMID: 19923322
20.  Substrate and Inhibitor Specificity of the Type II p21-Activated Kinase, PAK6 
PLoS ONE  2013;8(10):e77818.
The p21-activated kinases (PAKs) are important effectors of Rho-family small GTPases. The PAK family consists of two groups, type I and type II, which have different modes of regulation and signaling. PAK6, a type II PAK, influences behavior and locomotor function in mice and has an ascribed role in androgen receptor signaling. Here we show that PAK6 has a peptide substrate specificity very similar to the other type II PAKs, PAK4 and PAK5 (PAK7). We find that PAK6 catalytic activity is inhibited by a peptide corresponding to its N-terminal pseudosubstrate. Introduction of a melanoma-associated mutation, P52L, into this peptide reduces pseudosubstrate autoinhibition of PAK6, and increases phosphorylation of its substrate PACSIN1 (Syndapin I) in cells. Finally we determine two co-crystal structures of PAK6 catalytic domain in complex with ATP-competitive inhibitors. We determined the 1.4 Å co-crystal structure of PAK6 with the type II PAK inhibitor PF-3758309, and the 1.95 Å co-crystal structure of PAK6 with sunitinib. These findings provide new insights into the structure-function relationships of PAK6 and may facilitate development of PAK6 targeted therapies.
doi:10.1371/journal.pone.0077818
PMCID: PMC3810134  PMID: 24204982
21.  PAK1-mediated activation of ERK1/2 regulates lamellipodial dynamics 
Journal of cell science  2008;121(Pt 22):3729-3736.
Summary
PAK1 is a member of the p21-activated kinase (PAK) family of serine/threonine kinases that are activated by the Rho GTPases Rac and Cdc42 and are implicated in regulating morphological polarity, cell migration and adhesion. Here we investigate the function of PAK1 in cell motility using macrophages derived from PAK1-null mice. We show that CSF-1, a macrophage chemoattractant, transiently stimulates PAK1 and MAPK activation, and that MAPK activation is reduced in PAK1−/− macrophages. PAK1 regulates the dynamics of lamellipodium extension as cells spread in response to adhesion but is not essential for macrophage migration or chemotaxis towards CSF-1. Following adhesion, PAK1−/− macrophages spread more rapidly than wild-type macrophages and have more but less stable lamellipodia. ERK1/2 activity was reduced in PAK1−/− macrophages during adhesion, and inhibition of ERK1/2 activation in wild-type macrophages was sufficient to increase the spread area and mimic the lamellipodial dynamics of PAK1−/− macrophages. Together, these data indicate that PAK1 signals via ERK1/2 to regulate lamellipodial stability.
doi:10.1242/jcs.027680
PMCID: PMC2702775  PMID: 18940914
PAK1; ERK1/2; cell adhesion; cell migration; macrophages; Rho GTPases
22.  Elevated p21-Activated Kinase 2 Activity Results in Anchorage-Independent Growth and Resistance to Anticancer Drug-Induced Cell Death1 
Neoplasia (New York, N.Y.)  2009;11(3):286-297.
p21-Activated kinase 2 (PAK-2) seems to be a regulatory switch between cell survival and cell death signaling. We have shown previously that activation of full-length PAK-2 by Rac or Cdc42 stimulates cell survival, whereas caspase activation of PAK-2 to the proapoptotic PAK-2p34 fragment is involved in the cell death response. In this study, we present a role of elevated activity of full-length PAK-2 in anchorage-independent growth and resistance to anticancer drug-induced apoptosis of cancer cells. Hs578T human breast cancer cells that have low levels of PAK-2 activity were more sensitive to anticancer drug-induced apoptosis and showed higher levels of caspase activation of PAK-2 than MDA-MB435 and MCF-7 human breast cancer cells that have high levels of PAK-2 activity. To examine the role of elevated PAK-2 activity in breast cancer, we have introduced a conditionally active PAK-2 into Hs578T human breast cells. Conditional activation of PAK-2 causes loss of contact inhibition and anchorage-independent growth of Hs578T cells. Furthermore, conditional activation of PAK-2 suppresses activation of caspase 3, caspase activation of PAK-2, and apoptosis of Hs578T cells in response to the anticancer drug cisplatin. Our data suggest a novel mechanism by which full-length PAK-2 activity controls the apoptotic response by regulating levels of activated caspase 3 and thereby its own cleavage to the proapoptotic PAK-2p34 fragment. As a result, elevated PAK-2 activity interrupts the apoptotic response and thereby causes anchorage-independent survival and growth and resistance to anticancer drug-induced apoptosis.
PMCID: PMC2647731  PMID: 19242610
23.  Thymoquinone-induced conformational changes of PAK1 interrupt prosurvival MEK-ERK signaling in colorectal cancer 
Molecular Cancer  2014;13(1):201.
Background
Thymoquinone (TQ) was shown to reduce tumor growth in several cancer models both in vitro and in vivo. So far only a few targets of TQ, including protein kinases have been identified. Considering that kinases are promising candidates for targeted anticancer therapy, we studied the complex kinase network regulated by TQ.
Methods
Novel kinase targets influenced by TQ were revealed by in silico analysis of peptide array data obtained from TQ-treated HCT116wt cells. Western blotting and kinase activity assays were used to determine changes in kinase expression patterns in colorectal cancer cells (HCT116wt, DLD-1, HT29). To study the viability/apoptotic effects of combining the PAK1 inhibitor IPA-3 and TQ, crystal violet assay and AnnexinV/PI staining were employed. Interactions between PAK1 and ERK1/2 were investigated by co-immunoprecipitation and modeled by docking studies. Transfection with different PAK1 mutants unraveled the role of TQ-induced changes in PAK1 phosphorylation and TQ´s effects on PAK1 scaffold function.
Results
Of the 104 proteins identified, 50 were upregulated ≥2 fold by TQ and included molecules in the AKT-MEK-ERK1/2 pathway. Oncogenic PAK1 emerged as an interesting TQ target. Time-dependent changes in two PAK1 phosphorylation sites generated a specific kinase profile with early increase in pPAKThr212 followed by late increase in pPAKThr423. TQ induced an increase of pERK1/2 and triggered the early formation of an ERK1/2-PAK1 complex. Modeling confirmed that TQ binds in the vicinity of Thr212 accompanied by conformational changes in ERK2-PAK1 binding. Transfecting the cells with the non-phosphorylatable mutant T212A revealed an increase of pPAKThr423 and enhanced apoptosis. Likewise, an increase in apoptosis was observed in cells transfected with both the kinase-dead K299R mutant and PAK1 siRNA. Using structural modeling we suggest that TQ interferes also with the kinase domain consequently disturbing its interaction with pPAKThr423, finally inhibiting MEK-ERK1/2 signaling and disrupting its prosurvival function. pERK1/2 loss was also validated in vivo.
Conclusions
Our study shows for the first time that the small molecule TQ directly binds to PAK1 changing its conformation and scaffold function. Because TQ affects the central RAF/MEK/ERK1/2 pathway, the combination of TQ with targeted therapies is worth considering for future anticancer treatments.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-201) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-201
PMCID: PMC4158125  PMID: 25174975
Thymoquinone; PAK1; ERK1/2; Colorectal cancer; Scaffold function; Kinome analysis; Apoptosis
24.  PAK in Alzheimer disease, Huntington disease and X-linked mental retardation 
Cellular Logistics  2012;2(2):117-125.
Developmental cognitive deficits including X-linked mental retardation (XLMR) can be caused by mutations in P21-activated kinase 3 (PAK3) that disrupt actin dynamics in dendritic spines. Neurodegenerative diseases such as Alzheimer disease (AD), where both PAK1 and PAK3 are dysregulated, may share final common pathways with XLMR. Independent of familial mutation, cognitive deficits emerging with aging, notably AD, begin after decades of normal function. This prolonged prodromal period involves the buildup of amyloid-β (Aβ) extracellular plaques and intraneuronal neurofibrillary tangles (NFT). Subsequently region dependent deficits in synapses, dendritic spines and cognition coincide with dysregulation in PAK1 and PAK. Specifically proximal to decline, cytoplasmic levels of actin-regulating Rho GTPase and PAK1 kinase are decreased in moderate to severe AD, while aberrant activation and translocation of PAK1 appears around the onset of cognitive deficits. Downstream to PAK1, LIM kinase inactivates cofilin, contributing to cofilin pathology, while the activation of Rho-dependent kinase ROCK increases Aβ production. Aβ activation of fyn disrupts neuronal PAK1 and ROCK-mediated signaling, resulting in synaptic deficits. Reductions in PAK1 by the anti-amyloid compound curcumin suppress synaptotoxicity. Similarly other neurological disorders, including Huntington disease (HD) show dysregulation of PAKs. PAK1 modulates mutant huntingtin toxicity by enhancing huntingtin aggregation, and inhibition of PAK activity protects HD as well as fragile X syndrome (FXS) symptoms. Since PAK plays critical roles in learning and memory and is disrupted in many cognitive disorders, targeting PAK signaling in AD, HD and XLMR may be a novel common therapeutic target for AD, HD and XLMR.
doi:10.4161/cl.21602
PMCID: PMC3490962  PMID: 23162743
Alzheimer disease; curcumin; PAK; ROCK; signaling pathways; synapses
25.  p21-activated kinase 4 interacts with integrin αvβ5 and regulates αvβ5-mediated cell migration 
The Journal of Cell Biology  2002;158(7):1287-1297.
p21-activated kinase 1 (PAK1) can affect cell migration (Price et al., 1998; del Pozo et al., 2000) and modulate myosin light chain kinase and LIM kinase, which are components of the cellular motility machinery (Edwards, D.C., L.C. Sanders, G.M. Bokoch, and G.N. Gill. 1999. Nature Cell Biol. 1:253–259; Sanders, L.C., F. Matsumura, G.M. Bokoch, and P. de Lanerolle. 1999. Science. 283:2083–2085). We here present a novel cell motility pathway by demonstrating that PAK4 directly interacts with an integrin intracellular domain and regulates carcinoma cell motility in an integrin-specific manner. Yeast two-hybrid screening identified PAK4 binding to the cytoplasmic domain of the integrin β5 subunit, an association that was also found in mammalian cells between endogenous PAK4 and integrin αvβ5. Furthermore, we mapped the PAK4 binding to the membrane-proximal region of integrin β5, and identified an integrin-binding domain at aa 505–530 in the COOH terminus of PAK4. Importantly, engagement of integrin αvβ5 by cell attachment to vitronectin led to a redistribution of PAK4 from the cytosol to dynamic lamellipodial structures where PAK4 colocalized with integrin αvβ5. Functionally, PAK4 induced integrin αvβ5–mediated, but not β1-mediated, human breast carcinoma cell migration, while no changes in integrin cell surface expression levels were observed. In conclusion, our results demonstrate that PAK4 interacts with integrin αvβ5 and selectively promotes integrin αvβ5–mediated cell migration.
doi:10.1083/jcb.200207008
PMCID: PMC2173231  PMID: 12356872
cell motility; cell signaling; integrin; lamellipodia; p21-activated kinase

Results 1-25 (1021131)