Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

PAKs

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101590

Synonyms

Historical Background

PAKs are a family of protein kinases that were first identified in a screen for proteins that interact with the small G proteins Rac1 and Cdc42. Since these have a molecular weight of 21kd, this kinase was named p21-activated kinase (Manser et al. 1994). PAKs are widely conserved and found in yeast as well as in Drosophila and mammals. They are divided into two groups, group I, which consists of PAK1 (α PAK), PAK2 (γ PAK), and PAK3 (β PAK), and group II which consists of PAK4, PAK5, and PAK6 (Rane and Minden 2014). PAK4, which was identified from a PCR screen with degenerate primers based on the PAK2 kinase domain (Abo et al. 1998; Cotteret and Chernoff 2006), is the founding member of group II PAKs. PAK6 was identified as an androgen receptor (AR)-interacting protein in a yeast two-hybrid screen (Yang et al. 2001). The name PAK7 was retired in 2016, since it is no longer considered a separate protein from PAK 5.

PAK Expression

Group I PAKs share a high level of sequence homology, but they all have different tissue-specific expression patterns. PAK2 is found in all tissues (Ng et al. 2010), whereas PAK 1 and 3 have more restricted expression patterns. PAK1 is found in several tissues including mammary gland, muscle, and spleen (Manser et al. 1995), and all of the group I PAKs are highly expressed in the nervous system. PAK5 expression is enriched in the brain (Timm et al. 2006). PAK4 and PAK6 are expressed in prostate and testis, and PAK1 and PAK4 are expressed in the colon (Bokoch 2003). Note that multiple PAKs are often expressed in the same tissues.

PAK Structure

All PAKs have a C-terminal kinase domain and an N-terminal regulatory domain. The regulatory domains of all PAKs contain a p21-binding domain (PBD) which binds to activated Rac or Cdc42 and several proline-rich regions that serve as docking sites for SH3 domain-containing proteins (Fig. 1). The kinase domains of PAKs in group I are at least 93% homologous and about 54% homologous to members of the other group (Dummler et al. 2009). The PBD overlaps with a domain that plays an important regulatory role, called the autoinhibitory domain (AID) (Pirruccello et al. 2006; Zhao and Manser 2012). The group II PAKs were thought not to contain autoinhibitory domains at first, but later found to contain sequences related to AIDs (Ching et al. 2003; Ha et al. 2012). The group II PAKs, like the group I PAKs, also have N-terminal PBD and carboxyl-terminal kinase domains, but they do not contain the other conserved domains found in the group I PAKs. Furthermore, the PBD and kinase domains of the group II PAKs have only approximately 50% identity with those of group I, and the regulatory domains outside of the PBD are completely different from the group I PAKs.
PAKs, Fig. 1

Structures of PAKs. Group I PAKs have an autoinhibitory domain (AID) overlapping the PBD, while the group II PAKs have a related sequence adjacent to the PBD

PAK Activation

Group I and group II PAKs are regulated by different mechanisms (Radu et al. 2014). Models for the regulation of the two groups of PAKs are shown in Fig. 2. The kinases in group I function as dimers, where the AID binds in trans to the PAK catalytic domain on the dimerizing PAK. This interaction prevents autophosphorylation at the activation loop (A-loop) and the subsequent activation of PAK’s kinase activity (Lei et al. 2000). When Cdc42/Rac binds to PBD, the interaction between the AID and the dimerizing PAK is disrupted. This leads to a conformational change to the PAK and causes the PAK to become a monomer, which subsequently becomes autophosphorylated on the A-loop and several other sites, and activated (Buchwald et al. 2001).
PAKs, Fig. 2

Models representing the activation mechanism of PAKs. (A) Activation of the group I PAKs: The AID of one PAK binds the kinase domain of the dimerizing PAK in dimers and inactivates the group I PAKs. Cdc42 or Rac binding to PBD disrupts the inhibition, resulting in autophosphorylation and kinase activation. (B) Two different activation models of group II PAKs: In the first model (a), AID binding to the kinase domain of the monomeric PAK, in trans, inhibits its activation. Upon Cdc42 binding, PAKs take on an active conformation. In the second model (b), the autoinhibitory pseudosubstrate (PS) contains the RPKP sequence which can be recognized by the kinase domain, leading to the inhibition of PAKs. Binding with Cdc42 relocalizes PAK within the cell, and proteins containing SH3 domains, such as Src, subsequently activate PAK by competing with the PAK kinase domain, for interacting with the PS domain (modified from (Zhao and Manser 2012; Rane and Minden 2014))

Group II PAKs are activated by completely different mechanisms than the group I PAKs, as they function as monomers rather than dimers. A difference is that group II PAKs are constitutively autophosphorylated at the A-loop, even in quiescent cells. The group II PAK becomes active when the AID allosterically modifies the constitutively phosphorylated kinase rather than regulating A-loop autophosphorylation. There are two controversial models on how the AID-like domains function in group II PAK activation. The prevailing model is that the AID binds to the PAK catalytic domain in cis, which keeps the kinase in an inactive conformation, even though it is constitutively phosphorylated. When GTP-bound Cdc42 binds the PBD, this interaction is disrupted, leading to a conformational change which results in PAK activation (Baskaran et al. 2012).

The second model was proposed for PAK4, though PAK5 and PAK6 could operate by a similar mechanism. This model involves an autoinhibitory pseudosubstrate (PS) adjacent to the PBD like the AID in group I PAKs. According to the model, the PS is recognized by the PAK kinase domain, leading to an interaction between the two domains. The PS also has a proline-rich region that would be disrupted when the PS domain binds to proteins that contain SH3 domains, resulting in relief of autoinhibition. The activation process goes into two steps. First, Cdc42 or Rac binds to the PAK4 PBD. This would affect the localization of PAK, presumably bringing it to cellular regions containing other activating proteins and substrates. Upon relocalization, the second signal involves binding by SH3 domain-containing proteins, which relieves autoinhibition and activates the kinase. In fact, PAK4 can be activated by the Src SH3 domain, thus supporting this model (Ha et al. 2012).

Although group II PAKs were not thought to dimerize, as discussed above, a very recent study revealed that PAK5 is dimeric. The central region of PAK5 (residues 109–420) can promote self-association, and an elevated activity, but has no effect on activation loop phosphorylation. PAK5 self-association interferes with AID binding to the catalytic domain, which maintains its high activity (Tabanifar et al. 2016).

GTPase-Independent Activation

There are also ways to activate PAKs independent of GTPase binding. For example, PAK-interacting exchange factor (PIX), which is a guanine nucleotide exchange factor (gef) for Rac, binds to PAK through an SH3 interaction and recruits it to focal complexes. PIX also interacts with the G protein-coupled receptor kinase-interacting target (GIT1), and PAK1 is found in a trimeric complex with PIX/GIT1. Both PIX and GIT1 have key roles in targeting PAK1 to focal complexes. Activation can occur independently of Cdc42/Rac binding via GIT1 (Loo et al. 2004). Similar to what is seen at focal complexes, recruiting PAK1 to the centrosome activates PAK, which is independent of Cdc42 or Rac, but is dependent on centrosome integrity. Also, the cyclin-dependent kinases Cdk1 and Cdk5 phosphorylate PAK1 at Thr212 (Banerjee et al. 2002; Thiel et al. 2002). PDK1 can activate PAK1 by direct phosphorylation of Thr423 (King et al. 2000), and the adaptor proteins Nck and Grb2 bind proline-rich regions near the N-terminus of PAK and can activate PAK by directing it to receptor tyrosine kinases at the cell membrane (Bokoch et al. 1996; Galisteo et al. 1996; Lu et al. 1997). PAKs can also be activated by sphingolipids. Most of these GTPase-independent mechanisms have only been documented for group I PAKs.

Group II PAKs can also be regulated by other mechanisms that may be GTPase independent. In MDCK epithelial cells, PAK4 is activated by hepatocyte growth factor (HGF). HGF activation of PAK4 is dependent on PI3 kinase. Once cells are stimulated with HGF, PAK4 localizes to the cell periphery. In turn, PAK4 modulates cell adhesion and the organization of the cytoskeleton (Wells et al. 2002). PAK4 also binds to the cytoplasmic domain of the keratinocyte growth factor (KGF) receptor in a transformed kidney cell line, and it has important roles in cell survival pathways downstream to KGF (Lu et al. 2003). Finally, PAK6 plays a role in androgen receptor signaling in a pathway that is independent of the Rho GTPases.

PAK Signals and Substrates

There are numerous proteins that Pak phosphorylates, and consensus phosphorylation sequences for both group I and group II have been determined (Rennefahrt et al. 2007). The signaling pathways can be divided into four categories, kinase cascades, cytoskeletal, cell cycle, and cell survival. Examples of kinase cascades include ERK, in which Pak phosphorylates two upstream kinases c-Raf and Mek. The cytoskeletal cascade is primarily driven by LIMK, which phosphorylates cofilin. Cdk1 phosphorylates Pak. Pak kinases also play a key role in cell survival, as inhibitors of Pak cause apoptosis. Pak plays an integral role in Akt signaling, but the effect may be primarily through a scaffold role, and various reports have placed Pak upstream or downstream of Akt (Tang et al. 2000; Higuchi et al. 2008; Kim et al. 2011; Chow et al. 2012).

PAK Signaling in Cancer

PAKs and Rho GTPases have important roles in normal development, regulating cell survival, proliferation, cytoskeletal organization, and migration (Kelly and Chernoff 2012). Most interest in understanding the role of PAKs in human disease has focused on cancer. Multiple PAK family members maintain cell transformation by stimulating signaling pathways leading to proliferation, survival, motility, and angiogenesis (Fig. 3). PAK family members play a central role in cancer progression through the integration of cancer-promoting signals from cell membrane receptors as well as a key nexus-modifier of complex, cytoplasmic signaling networks. PAKs regulate several cell signaling pathways controlling tumor cell growth and survival including MAPK/Erks (Tang et al. 1997), p53 (Murray et al. 2010), NFκB (Frost et al. 2000), Smad (Lee et al. 2011), and STAT3 (Teng et al. 2009). In some cases, the relationship between PAKs with these signaling pathways has been established, while in other cases the direct connection with PAK has yet to be determined. The Erk, NFκB, and more recently p53 pathways are the best documented examples of PAK regulation of cancer signaling pathways.
PAKs, Fig. 3

PAK signaling in cancer. PAKs are effectors of Rac/Cdc42 and play a key role in some of cancers to stimulate proliferation, resist cell death, promote cell invasion and metastasis, and induce angiogenesis. PAKs can regulate cell proliferation through the Raf/Mek pathway. Cell motility can be affected by PAK phosphorylation of cytoskeletal targets, such as LIMK, which phosphorylates cofilin. PAK1 also phosphorylates Bad directly and indirectly via Raf-1, thus promoting cell survival by anti-apoptosis. NFκB regulated by PAK indirectly promotes cell survival

PAKs are not frequently mutated in human cancer; however, overexpression and gene amplification are commonly seen in cancer. PAK1 and PAK4 are the PAKs that are most strongly associated with cancer. Both PAK1 and PAK4 are localized to genomic regions that are frequently amplified in cancer cells (Ong et al. 2011; Ye and Field 2012). While gene amplification is one way that Pak proteins become overproduced in cancer, there are also other mechanisms leading to PAK overexpression. Here, we address PAK in several specific tumors for which a role has been established including breast, neurofibromatosis 1/2, colon, ovarian, pancreatic, and lung.

Breast and ovarian cancer. Breast cancers overexpress or hyperactivate PAK1 (Balasenthil et al. 2004; Ong et al. 2011), and expression levels correlate with invasiveness and increased survival. Several signaling pathways such as Erk and MET, NFκB, BAD, and estrogen receptor α (ER α) are activated by PAK1 as breast cancer progresses (Wang et al. 2002; Rayala et al. 2006; Friedland et al. 2007). PAK4 is also frequently overexpressed in breast cancer (Callow et al. 2002; Liu et al. 2008). Like PAK1, PAK4 is often found in its wild-type form in tumors (Chen et al. 2008; Kimmelman et al. 2008). PAK is activated through pathways that are important for breast cancer growth. Growth factors such as prolactin and the oncogene human epidermal growth factor receptor 2 (HER2 or ErbB2) can activate MAPK signaling pathway through PAK1. The prolactin receptor (PRL-R) can initiate and sustain Erk1/2 signaling via the PI3K-dependent Rac/PAK pathway. PAK also regulates survival and motility signals in breast cancer. In ovarian cancer, Pak is often amplified, and these tumors are more sensitive to Pak inhibition (Prudnikova et al. 2016).

Neurofibromatosis. Neurofibromatosis types 1 and 2 (NF1 and NF2) are dominantly inherited autosomal diseases caused by loss-of-function mutations in the tumor suppressor genes NF1 and NF2, respectively. PAK1 is important for the malignant growth in both types of neurofibromatosis. Deletion of either gene leads to abnormal activation of PAK1 through different mechanisms, though NF1 and NF2 are genetically and clinically distinct diseases. In NF1, PAK1 is activated through the Ras pathway. The product of the NF1 gene neurofibromin, a GTPase-activating protein (GAP), is widely expressed in a range of tissues but at high levels in the nervous system. Neurofibromin can increase the intrinsic GTPase activity of Ras. Consequently, loss of neurofibromin increases levels of activated GTP-bound Ras, which activates oncogenic pathways, including the MAPK cascade and PI3K. Downstream signals of PI3K activate PAK via Rac and Cdc42. While NF1 activates PAK through effector pathways, NF2 interacts directly with PAK1 through phosphorylation of Merlin, the product of the NF2 gene. Merlin also associates with inactive PAK and prevents its activation, perhaps by competing with Rac (Kissil et al. 2003; Hirokawa et al. 2004). PAK is a major player underlying Schwann cell transformation and an attractive target for therapeutics in both NF1 and NF2. There are multiple signaling pathways that PAK regulates in Schwann cells, and the signals may differ between NF1 and NF2.

Colon cancer. In 70% of colon cancer samples, PAK1 is overexpressed, which is correlated with several signals including, Wnt, Erk, and Akt activation. Downregulation of PAK1 decreases cell proliferation, migration/invasion, and survival. Besides PAK1, PAK4 and PAK5 have also been implicated in colon cancer cell transformation through expression studies as well as functional studies where they regulate cell adhesion and migration (Carter et al. 2004).

Pancreatic cancer. The role of PAKs in the development and progression of pancreatic cancer is largely unknown, although it has been shown that pancreatic tumors may have upregulated expression of PAK1 and PAK4. PAK1 is overexpressed in pancreatic cancer tissues compared with adjacent normal tissue (Jagadeeshan et al. 2015). Chauhan and colleagues found that MUC13, a transmembrane mucin, was overexpressed in pancreatic cancer, and this correlated with increased PAK1 expression and activity (Chauhan et al. 2012). Additionally, the PAK4 gene is amplified in approximately 20% of pancreatic cancer patients, and pancreatic tumors have increased PAK4 kinase activity. Although their role has not been completely elucidated, the available evidence shows that PAK1 and PAK4 do play a role in pancreatic cancer (Yeo et al. 2014).

Lung cancer. Lung cancer, although not as well established as other cancers, is emerging as a tumor depends on PAK1 signaling. A mouse model for Ras-induced lung cancers is highly sensitive to Rac inhibition, suggesting that lung cancers may be dependent on PAK (Kissil et al. 2007). PAK1 is expressed strongly in the nucleus and cytoplasm of squamous non-small cell lung carcinomas (NSCLCs). Finally, selective inhibition of PAK1 but not PAK2 delayed cell-cycle progression in vitro and in vivo.

PAK Inhibitors

Because of the central role of PAKs in controlling cytoskeletal organization, cell growth, and cell survival, in the migration in human cancers, there has been considerable interest in developing PAK inhibitors. Inhibitors have been developed by several companies, one of which briefly entered clinical trials but was withdrawn because of poor bioavailability (Murray et al. 2010). The structure of PAK now guides the design of specific PAK inhibitors (Field and Manser 2012; Jha and Strauss 2012). Because of their large and flexible ATP-binding cleft, PAKs, particularly group I PAKs, are difficult to drug; hence, few PAK inhibitors with satisfactory kinase selectivity and drug-like properties have been reported to date (Rudolph et al. 2015). However, several different inhibitors have been generated. IPA-3 (p21-activated kinase inhibitor 3), for example, is an allosteric inhibitor binding to the regulatory domain of PAK1 and is specific for the group I PAKs (Deacon et al. 2008). Peptide inhibitors have also been generated, including the PAK1 AID and TAT-PAK18 (Hashimoto et al. 2010). PAK1 shRNA inhibits cell proliferation, suggesting that shRNA may be another strategy for blocking the PAKs (Yi et al. 2008). FL172 (Maksimoska et al. 2008), FRAX1036 (Licciulli et al. 2013), FRAX597 (Ong et al. 2015), and OSU-03012 (Porchia et al. 2007) are examples of kinase inhibitors with some specificity toward the group I PAKs.

PF3758309 was designed specifically to block the PAK4 kinase activity, but was found to be broadly inhibitory toward both group I and group II PAKs and also other kinases, including AMPK (AMP-dependent kinase) and RSK (ribosomal S6 kinase). A survey with PF3758309 of 92 tumor cell lines derived from colorectal and non-small cell lung cancer and pancreatic and breast tumors found that 46% exhibited IC50 values less than 10 nM. It is encouraging that so many tumors respond to PAK inhibitors. PF3758309 entered clinical trials but did not progress beyond phase I trials (Murray et al. 2010). A recent analysis found that Pak inhibitors of diverse structural classes cause acute cardiotoxicity, perhaps because of the role that Pak2 plays in circulation (Rudolph et al. 2016). There is hope that this cardiotoxicity can be overcome by designing more specific inhibitors that bind to an allosteric site found only on Pak1, so these may not target Pak2 (Karpov et al. 2015).

Summary

Pak kinases are activated by many cell signaling pathways and play key roles in motility, proliferation, and survival. Several comprehensive reviews discuss Pak function and signals in depth, including an entire issue of Cellular Logistics (Field and Manser 2012; Ye and Field 2012; Radu et al. 2014; Rane and Minden 2014). Of practical interest are the signals central to cancer. Pak kinases are sometimes amplified themselves in cancer, but many of the key signals that Pak regulates are from the Ras oncogene. Since Ras is mutated in about 20% of tumors with mutations in Ras, it may respond to PAK inhibitors in addition to those in which PAK itself is amplified. Additionally, the synergies observed with PAK inhibitors and other drugs suggest that PAK inhibitors are likely to be most effective in combination with other treatments.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Systems Pharmacology and Translational Therapeutics, Perelman School of MedicineUniversity of PennsylvaniaPhiladelphiaUSA