Melanoma-associated mutants within the serine-rich domain of PAK5 direct kinase activity to mitogenic pathways.
ABSTRACT: The overexpression and hyperactivity of p21-activated serine/threonine kinases (PAKs) is known to facilitate tumorigenesis; however, the contribution of cancer-associated PAK mutations to tumor initiation and progression remains unclear. Here, we identify p21-activated serine/threonine kinase 5 (PAK5) as the most frequently altered PAK family member in human melanoma. More than 60% of melanoma-associated PAK5 gene alterations are missense mutations, and distribution of these variants throughout the protein coding sequence make it difficult to distinguish oncogenic drivers from passengers. To address this issue, we stably introduced the five most common melanoma-associated PAK5 missense mutations into human immortalized primary melanocytes (hMELTs). While expression of these mutants did not promote single-cell migration or induce temozolomide resistance, a subset of variants drove aberrant melanocyte proliferation. These mitogenic mutants, PAK5 S364L and D421N, clustered within an unstructured, serine-rich domain of the protein and inappropriately activated ERK and PKA through kinase-independent and -dependent mechanisms, respectively. Together, our findings establish the ability of mutant PAK5 to enhance PKA and MAPK signaling in melanocytes and localize the engagement of mitogenic pathways to a serine-rich region of PAK5.
Project description:Synaptic transmission is mediated by a complex set of molecular events that must be coordinated in time and space. While many proteins that function at the synapse have been identified, the signaling pathways regulating these molecules are poorly understood. Pak5 (p21-activated kinase 5) is a brain-specific isoform of the group II Pak kinases whose substrates and roles within the central nervous system are largely unknown. To gain insight into the physiological roles of Pak5, we engineered a Pak5 mutant to selectively radiolabel its substrates in murine brain extract. Using this approach, we identified two novel Pak5 substrates, Pacsin1 and Synaptojanin1, proteins that directly interact with one another to regulate synaptic vesicle endocytosis and recycling. Pacsin1 and Synaptojanin1 were phosphorylated by Pak5 and the other group II Paks in vitro, and Pak5 phosphorylation promoted Pacsin1-Synaptojanin1 binding both in vitro and in vivo. These results implicate Pak5 in Pacsin1- and Synaptojanin1-mediated synaptic vesicle trafficking and may partially account for the cognitive and behavioral deficits observed in group II Pak-deficient mice.
Project description:We have characterized a new member of the mammalian PAK family of serine/threonine kinases, PAK5, which is a novel target of the Rho GTPases Cdc42 and Rac. The kinase domain and GTPase-binding domain (GBD) of PAK5 are most closely related in sequence to those of mammalian PAK4. Outside of these domains, however, PAK5 is completely different in sequence from any known mammalian proteins. PAK5 does share considerable sequence homology with the Drosophila MBT protein (for "mushroom body tiny"), however, which is thought to play a role in development of cells in Drosophila brain. Interestingly, PAK5 is highly expressed in mammalian brain and is not expressed in most other tissues. We have found that PAK5, like Cdc42, promotes the induction of filopodia. In N1E-115 neuroblastoma cells, expression of PAK5 also triggered the induction of neurite-like processes, and a dominant-negative PAK5 mutant inhibited neurite outgrowth. Expression of activated PAK1 caused no noticeable changes in these cells. An activated mutant of PAK5 had an even more dramatic effect than wild-type PAK5, indicating that the morphologic changes induced by PAK5 are directly related to its kinase activity. Although PAK5 activates the JNK pathway, dominant-negative JNK did not inhibit neurite outgrowth. In contrast, the induction of neurites by PAK5 was abolished by expression of activated RhoA. Previous work has shown that Cdc42 and Rac promote neurite outgrowth by a pathway that is antagonistic to Rho. Our results suggest, therefore, that PAK5 operates downstream to Cdc42 and Rac and antagonizes Rho in the pathway, leading to neurite development.
Project description:P21-activated kinase 5 (PAK5), also termed PAK7, is one of the six members of the PAK family of serine/threonine kinases, which are downstream effectors in several cancer signaling pathways. PAK5 promotes neural outgrowth, contributes to microtubule stability and induces resistance to apoptosis. However, the clinical importance of PAK5 in gastric cancer has not been comprehensively investigated. In the present study, PAK5 expression was evaluated in gastric cancer tissue samples. Furthermore, the associations between high expression of PAK5, and clinicopathological features and prognosis were examined. PAK5 expression in primary gastric cancer specimens resected from 279 patients who underwent gastrectomy at the Tokyo Medical and Dental University Hospital was evaluated using immunohistochemistry. Of the 279 patients, 44 (15.8%) exhibited high PAK5 expression, which was significantly associated with the differentiated pathological type (differentiated vs. undifferentiated; P<0.001), depth of tumor invasion (T1 vs. T2-T4; P<0.001), lymph node metastasis (N0 vs. N1-N3; P<0.001), presence of distant metastasis or recurrence (present vs. absent; P=0.038), advanced tumor stage (I vs. II-IV; P=0.001) and worse disease-specific survival (P=0.013). In stage I-III disease, 38/254 (15.0%) patients exhibited high PAK5 expression, and high expression of PAK5 was significantly associated with relapse-free interval (P=0.044). PAK5 may serve an important role in tumor progression and influence the outcome of patients with gastric cancer.
Project description: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.
Project description:The p21-activated kinases (PAKs) are a family of six serine/threonine kinases that act as key effectors of RHO family GTPases in mammalian cells. PAKs are subdivided into two groups: type I PAKs (PAK1, PAK2, and PAK3) and type II PAKs (PAK4, PAK5, and PAK6). Although these groups are involved in common signaling pathways, recent work indicates that the two groups have distinct modes of regulation and have both unique and common substrates. Here, we review recent insights into the molecular level details that govern regulation of type II PAK signaling. We also consider mechanisms by which signal transduction is regulated at the level of substrate specificity. Finally, we discuss the implications of these studies for clinical targeting of these kinases.
Project description:The six serine/threonine kinases in the p21-activated kinase (PAK) family are important regulators of cell adhesion, motility and survival. PAK6, which is overexpressed in prostate cancer, was recently reported to localize to cell-cell adhesions and to drive epithelial cell colony escape. Here we report that PAK6 targeting to cell-cell adhesions occurs through its N-terminus, requiring both its Cdc42/Rac interactive binding (CRIB) domain and an adjacent polybasic region for maximal targeting efficiency. We find PAK6 localization to cell-cell adhesions is Cdc42-dependent, as Cdc42 knockdown inhibits PAK6 targeting to cell-cell adhesions. We further find the ability of PAK6 to drive epithelial cell colony escape requires kinase activity and is disrupted by mutations that perturb PAK6 cell-cell adhesion targeting. Finally, we demonstrate that all type II PAKs (PAK4, PAK5 and PAK6) target to cell-cell adhesions, albeit to differing extents, but PAK1 (a type I PAK) does not. Notably, the ability of a PAK isoform to drive epithelial colony escape correlates with its targeting to cell-cell adhesions. We conclude that PAKs have a broader role in the regulation of cell-cell adhesions than previously appreciated.
Project description:p21-activated kinases have been classified into two groups based on their domain architecture. Group II PAKs (PAK4-6) regulate a wide variety of cellular functions, and PAK deregulation has been linked to tumor development. Structural comparison of five high-resolution structures comprising all active, monophosphorylated group II catalytic domains revealed a surprising degree of domain plasticity, including a number of catalytically productive and nonproductive conformers. Rearrangements of helix alphaC, a key regulatory element of kinase function, resulted in an additional helical turn at the alphaC N terminus and a distortion of its C terminus, a movement hitherto unseen in protein kinases. The observed structural changes led to the formation of interactions between conserved residues that structurally link the glycine-rich loop, alphaC, and the activation segment and firmly anchor alphaC in an active conformation. Inhibitor screening identified six potent PAK inhibitors from which a tri-substituted purine inhibitor was cocrystallized with PAK4 and PAK5.
Project description:p21-activated kinases (PAKs) are multifunctional effectors of Rho GTPases, which are associated with cytoskeletal organization, cellular morphogenesis, migration and survival. PAKs are overactive in a number of tumor tissues and have attracted attention as a potential target for cancer therapy. In the present study, PAK5 levels were analyzed in primary osteosarcoma (OS) samples (n=65) using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) methods. In the primary OS tissue, increased PAK5 expression (IHC score >2, n=37) was associated with significantly decreased overall survival (P=0.036) compared with decreased PAK5 expression (IHC score ?2, n=28). PAK5 expression was identified to be significantly associated with metastasis (P=0.010). The lung is the most common metastasis site for OS. In addition, the level of PAK5 in lung metastasis tissue (n=13) was detected using RT-qPCR and IHC methods. PAK5 expression was increased in lung metastasis tissue compared with in primary OS samples. PAK5 was silenced using short hairpin RNA in OS cell lines. Wound healing, migration and nude mice model assay results consistently demonstrated that PAK5 knockdown was able to significantly inhibit OS migration. In PAK5-knockdown cells, the alteration in the expression of a number of metastasis-associated factors, including epithelial cadherin, vimentin, fibronectin and matrix metalloproteinase 2 (MMP2), was analyzed. Only MMP2 expression was decreased significantly (P<0.05). The expression level of MMP2 was analyzed in primary OS tissue and lung metastasis tissue using RT-qPCR and IHC methods. Expression of MMP2 was identified to be associated with expression of PAK5. The results of the present study suggest that PAK5 promotes OS cell migration and that PAK5 expression may be used to predict lung metastasis.
Project description:The ?-Melanocyte Stimulating Hormone (?MSH)/Melanocortin-1 receptor (MC1R) interaction promotes melanogenesis through the cAMP/PKA pathway. The direct induction of this pathway by Forskolin (FSK) is also known to enhance melanocyte proliferation. ?MSH acts as a mitogenic agent in melanocytes and its effect on proliferation of melanoma cells is less known. We previously identified the ?MSH/Peroxisome Proliferator Activated Receptor (PPAR?) pathway as a new pathway on the B16-F10 mouse melanoma cell line. ?MSH induced the translocation of PPAR? into the nucleus as an active transcription factor. This effect was independent of the cAMP/PKA pathway and was mediated by the activation of the PI(4,5)P2/PLC pathway, a pathway which we have described to be triggered by the ?MSH-dependent MC1R stimulation. Moreover, in the same study, preliminary experiments showed that mouse melanoma cells responded to ?MSH by reducing proliferation and that PPAR? was involved in this effect. Due to its key role in the control of cell proliferation, PPAR? agonists are used in therapeutic models for different forms of cancer, including melanoma. The purpose of this study was: (a) to confirm the different proliferative behavior in response to ?MSH in healthy and in melanoma condition; (b) to verify whether the cAMP/PKA pathway and the PLC/PPAR? pathway could exert an antagonistic function in the control of proliferation; (c) to deepen the knowledge of the molecular basis responsible for the down-proliferative response of melanoma cells after exposure to ?MSH.We employed B16-F10 cell line, a human melanoma cell line (Mel 13) and two primary cultures of human melanocytes (NHM 1 and NHM 2, respectively), all expressing a wild type MC1R and responding to the ?MSH in terms of pigmentation. We evaluated cell proliferation through: a) cell counting, b) cell cycle analysis c) protein expression of proliferation modulators (p27, p21, cyclin D1 and cyclin E).The ?MSH acted as a mitogenic agent in primary cultures of human melanocytes, whereas it determined a slow down of proliferation in melanoma cell lines. FSK, as an inducer of the cAMP/PKA pathway, reproduced the ?MSH mediated effect on proliferation in NHMs but it did not mimic the ?MSH effect on proliferation in B16-F10 and Mel 13 melanoma cell lines. Meanwhile, 3 M3-FBS (3 M3), as an inducer of PI(4,5)P2/PLC pathway, reproduced the ?MSH proliferative effect. Further experiments, treating melanoma cell lines with ?MSH in the presence/absence of GW9662, as an inhibitor of PPAR?, confirmed the key role of this transcription factor in decreasing cell proliferation in response to the hormone exposure.In both melanoma cell lines, ?MSH determined the reduction of proliferation through the PI(4,5)P2/PLC pathway, employing PPAR? as an effector element. These evidence could offer perspectives for new therapeutic approaches for melanoma.
Project description:MARK/Par-1 is a kinase involved in development of embryonic polarity. In neurons, MARK phosphorylates tau protein and causes its detachment from microtubules, the tracks of axonal transport. Because the target sites of MARK on tau occur at an early stage of Alzheimer neurodegeneration, we searched for interaction partners of MARK. Here we report that MARK2 is negatively regulated by PAK5, a neuronal member of the p21-activated kinase family. PAK5 suppresses the activity of MARK2 toward its target, tau protein. The inhibition requires the binding between the PAK5 and MARK2 catalytic domains, but does not require phosphorylation. In transfected Chinese hamster ovary (CHO) cells both kinases show a vesicular distribution with partial colocalization on endosomes containing AP-1/2. Although MARK2 transfected alone destabilizes microtubules and stabilizes actin stress fibers, PAK5 keeps microtubules stable through the down-regulation of MARK2 but destabilizes the F-actin network so that stress fibers and focal adhesions disappear and cells develop filopodia. The results point to an inverse relationship between actin- and microtubule-related signaling by the PAK5 and MARK2 pathways that affect both cytoskeletal networks.