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Review
. 2022 Apr;97(2):615-639.
doi: 10.1111/brv.12815. Epub 2021 Nov 22.

Group I PAKs in myelin formation and repair of the central nervous system: what, when, and how

Yan Wang  1 , Fuzheng Guo  1
Affiliations
Review

Group I PAKs in myelin formation and repair of the central nervous system: what, when, and how

Yan Wang et al. Biol Rev Camb Philos Soc. 2022 Apr.

Abstract

p21-activated kinases (PAKs) are a family of cell division control protein 42/ras-related C3 botulinum toxin substrate 1 (Cdc42/Rac1)-activated serine/threonine kinases. Group I PAKs (PAK1-3) have distinct activation mechanisms from group II PAKs (PAK4-6) and are the focus of this review. In transformed cancer cells, PAKs regulate a variety of cellular processes and molecular pathways which are also important for myelin formation and repair in the central nervous system (CNS). De novo mutations in group I PAKs are frequently seen in children with neurodevelopmental defects and white matter anomalies. Group I PAKs regulate virtually every aspect of neuronal development and function. Yet their functions in CNS myelination and remyelination remain incompletely defined. Herein, we highlight the current understanding of PAKs in regulating cellular and molecular pathways and discuss the status of PAK-regulated pathways in oligodendrocyte development. We point out outstanding questions and future directions in the research field of group I PAKs and oligodendrocyte development.

Keywords: demyelination; differentiation; multiple sclerosis (MS); myelination; oligodendrocyte progenitor cells (OPCs); oligodendrocytes (OLs); p21-activated kinases (PAKs); remyelination.

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Figures

Fig. 1.
Fig. 1.
Structure, activation, and homology among group I p21-activated kinases (PAKs). (A) Schematic diagram depicting the major domains and motifs for protein–protein interactions, major phosphorylation sites, and respective kinases using PAK1 as an example. AID, autoinhibitory domain; Akt/PKB, protein kinase B; Cdc2, cell division control 2; CRIB, Cdc42 and Rac1 interactive binding domain; Gβγ, G protein beta gamma; Grb2, growth factor receptor-bound protein 2; Nck, non-catalytic region of tyrosine kinase; p35/Cdk5,; PDK1, 3-phosphoinositide-dependent kinase 1; PIX, p21-interacting Rac-guanine exchange factor; PKG, cGMP-dependent protein kinase. (B) PAK1 is autoinhibited in trans and the binding of GTP-Cdc42 or GTP-Rac1 induces a conformational change, autophosphorylation at Ser144 and Thr423, and kinase activation. Fully activated PAK1 may undergo dephosphorylation by certain phosphatases (Wang & Wang, 2008; Ke et al., 2004) and convert to autoinhibited homodimers or be subject to proteasome-mediated degradation (Weisz Hubsman et al., 2007), thus down-regulating PAK activity. (C) Amino acid (aa) length and predicted molecular mass of PAK1–3. Sequence % homology was retrieved from Uniprot.org and calculated as the number of identical aa residues between species. (D) Amino acid sequences of human PAK1 and PAK2. Residues and numbers highlighted in red are the major autophosphorylation sites during kinase activation. PAK1 Lys299 and PAK2 Lys278 (highlighted in green) are ATP binding sites of the activated PAKs. PAK1 Thr212 (highlighted in blue) is a phosphorylated site present only in PAK1. Mutation of the lysine residue to other residues, for example to arginine (K299R), prevents ATP binding and creates a kinase-inactive PAK1 (kinase-dead PAK1). Therefore, expression of K299R-PAK1 plasmids, in vivo or in vitro, provides a dominant-negative approach to probe the role of PAK1 kinase activity.
Fig. 2.
Fig. 2.
Expression of the p21-activated kinases PAK1, PAK2, and PAK3 in the body, central nervous system (CNS), and in oligodendroglial-lineage cells. (A) Relative levels of human PAK1–3 protein in different organs. Data adapted from the Human Protein Atlas (www.proteinatlas.org). (B) Relative levels of mouse PAK1–3 messenger RNA (mRNA) in different brain cells. Values are means ± standard deviations. Data adapted from the brain RNA-sequencing (RNA-seq) database (www.brainrnaseq.org) (Zhang et al., 2014). FPKM, fragments per kilobase of transcript per million mapped reads; myelinating OL, myelinating oligodendrocytes identified by myelin oligodendrocyte glycoprotein (MOG) expression; NFO, newly formed oligodendrocytes identified by galactosylceramidase (GalC) expression; OPC, oligodendrocyte progenitor cells. (C) Relative levels of mouse PAK1–3 mRNA in different maturation stages from OPC to mature oligodendrocytes stage 6 (MO6) (from left to right). COP, differentiation-committed OPC; NFOL, newly formed oligodendrocytes; MFOL, myelin-forming oligodendrocytes. The oligodendroglial lineage-specific marker myelin regulatory factor (MYRF) (Emery et al., 2009) serves as a control against which the relative level of PAK1–3 can be compared. Data adapted from single-cell RNA-seq (www.linnarssonlab.org/oligodendrocytes) (Marques et al., 2016). The y-axis shows unique molecular identifier (UMI) counts.
Fig. 3.
Fig. 3.
p21-activated kinases (PAKs) and cytoskeleton in oligodendrocyte progenitor cells (OPCs) and oligodendrocytes (OLs). (A) Major steps of morphological differentiation from OPCs to myelinating OLs. (B) Simplified diagram depicting actin and microtubule (MT) cytoskeleton distribution in OL processes which are analogues to neuronal growth cones. The F-actin network is concentrated at the leading edge of the growth cone-like structures whereas the MT network is distributed in parallel orientation along the processes. Non-muscle myosin II participates in actin stress fibre formation, which provides contractile forces for migrating mammalian cells. (C) Primary OPC culture and validation of OPC differentiation (Zhang et al., 2018). D1–D4, days 1–4; DM, differentiation medium; GM, growth medium; MBP, myelin basic protein. (D) Representative image showing MTs revealed by α-tubulin immunostaining in a D2 immature OL. Image adapted from Lang et al. (2013). Scale bar, 10μm. (E–G) Actin cytoskeleton (indicated by phalloidin staining; red) and PAK1 distribution (green) in OPCs (E) D2 immature OLs (F), and D4 mature OLs (G). DAPI, 4,6-diamidine-2-phenylindole is blue-fluorescent DNA stain. Scale bars: 10μm. In E, OPC#4 is shown at a higher magnification in the images on the right. Dotted outline in the second image from the left indicates the outermost rim of the F-actin network. Arrows point to the leading filopodial-like spikes positive for F-actin. Arrowheads point to the outermost edge of the F-actin-rich lamellipodia-like protrusions. In F, the boxed area is shown at a higher magnification in the lower panels. The arrowheads indicate the F-actin+ filopodia-like spikes at the end of oligodendroglial processes that appear negative for PAK1. (G) Distribution of PAK1 and F-actin in D4 mature OLs. F-actin is localized in distal processes and myelin sheets whereas PAK1 is restricted to the primary and secondary processes and cell bodies. F-actin is often concentrated in the F-actin-rich lamellipodia-like protrusions which are negative for PAK1 (arrowheads). The PAK1 antibody used was a validated antibody (#223849; Abcam) (Grebenova et al., 2019). Note that the Abcam PAK1 antibody #131522 previously used for immunostaining of primary OLs (Brown et al., 2021) was shown to recognize an unknown antigen but not endogenous PAK1 (Grebenova et al., 2019). (H) PAKs regulate cytoskeleton dynamics by phosphorylating and modulating the activity of their substrates. The best studied substrate is Lin-11, Islet-1, and Mec-3 domain kinase 1 (LIMK1), which is phosphorylated at residue T508 and subsequently activated by PAKs. Activated LIMK1 phosphorylates cofilin and inhibits the actin depolymerizing and severing activity of cofilin (Delorme et al., 2007). PAKs phosphorylate filamin (Barnes et al., 2003) and actin-related protein 2/3 complex 41 kDa subunit (p41/Arc) (Vadlamudi et al., 2004b), the regulatory component of the actin-related protein 2/3 complex (Arp2/3), to promote actin polymerization. PAKs regulate actin stress fibre formation by phosphorylating and inhibiting myosin light chain kinase (MLCK) (Goeckeler et al., 2000; Sanders et al., 1999). PAKs regulate MT dynamics by phosphorylating and modulating the activity of the MT-destabilizing protein stathmin (Wittmann et al., 2004) and the MT polymerizing protein tubulin cofactor B (TBCB) (Vadlamudi et al., 2005a).
Fig. 4.
Fig. 4.
p21-activated kinases (PAKs) regulate Wnt/β-catenin (A) and protein kinase B (Akt/PKB) (B) signalling pathways. (A) PAK activates the β-catenin-mediated signalling pathway both directly and indirectly. In the absence of upstream Wnt signalling, β-catenin undergoes rapid turnover, a proteasome-mediated degrading process involving the destruction complex consisting of adenomatous polyposis coli (Apc), Axin2, casein kinase I (Ck1), and glycogen synthesis kinase 3 (Gsk3). Ck1-mediated phosphorylation at Ser45 and subsequent Gsk3-mediated phosphorylation at Ser33/Ser37/Thr41 mark β-catenin for proteasome-mediated degradation. Kinase-active PAKs directly interact with and phosphorylate β-catenin at Ser663 and S675, both of which stabilize β-catenin and activate β-catenin/T cell factor (TCF)/lymphoid enhancer factor (LEF)-mediated signalling. PAKs also indirectly enhance β-catenin transcriptional activity by potentiating serine/threonine kinase Akt/PKB activation (Higuchi et al., 2008), which, in turn, phosphorylates β-catenin at Ser552 and increases its stability and transcriptional activity. (B) Reciprocal regulation between PAK and Akt/PKB signalling pathways. PAK1 is re-localized to the cell membrane by binding to adaptor proteins (such as non-catalytic region of tyrosine kinase, Nck) through its proline-rich PxxP motifs (see Fig. 1A) in response to growth factor-activated receptor tyrosine kinases (RTKs). PAK1 stimulates Akt/PKB through its kinase-independent scaffolding function by recruiting Akt/PKB from the cytosol to the cell membrane where Akt/PKB is activated by the membrane-associated 3-phosphoinositide-dependent protein kinase-1 (PDK1) (Higuchi et al., 2008). PAK1 also positively regulates Akt/PKB by directly phosphorylating Akt/PKB at Ser473 at the C-terminal regulatory domain (Mao et al., 2008), an essential phosphorylation event for Akt/PKB activation. Reciprocally, activated Akt/PKB disassociates PAK1 from the cell membrane by phosphorylating PAK1 at Ser21 (Zhou et al., 2003), a residue that was originally identified as an autophosphorylation site during PAK activation. Akt/PKB also promotes PAK activation (Tang et al., 2000) possibly through the mammalian target of rapamycin (mTOR)/P70S6 kinase signalling pathway (Ishida et al., 2007).
Fig. 5.
Fig. 5.
Regulation of rapidly accelerated fibrosarcoma 1 (Raf1)/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) (A) and Notch (B) signalling pathways by p21-activated kinases (PAKs). (A) PAKs positively and negatively regulate the Raf1/MEK/ERK signalling pathway in different contexts. PAK1 activates ERK signalling pathways by directly phosphorylating the upstream activator Raf1 at Ser338 and MEK at S298 and augmenting the kinase activity of Raf1 and MEK (Zang et al., 2002; Chaudhary et al., 2000; Shrestha et al., 2012; Coles & Shaw, 2002). PAK1 promotes ERK activation independent of its kinase activity and presumably by PAK’s scaffold function which recruits MEK to the cell membrane where MEK can be activated by membrane-bound activated Raf1 (Wang et al., 2013). PAK1 also inhibits ERK signalling by activating protein phosphatase 2A (PP2A) (Taglieri et al., 2011; Staser et al., 2013), a negative regulator of ERK activity (Miglietta et al., 2006; Van Kanegan et al., 2005). (B) Hypothetical model of PAK regulation of Notch signalling. Notch receptors are membrane-anchored proteins consisting of an extracellular ligand-binding domain and an intracellular domain (NICD, Notch intracellular domain). Notch ligands are membrane-associated glycoproteins (such as Jagged1, Delta, and F3/contactin) expressed in Notch signal-producing cells. PAK1 interacts with and phosphorylates the Notch repressor component SHARP (SMRT/HDAC1 associated repressor protein) at Ser3486 and Thr3568 within its repression domain, enhancing SHARP-mediated repression of Notch target genes (Vadlamudi et al., 2005b). PAK1 also controls Notch signalling through phosphorylating and activating integrin-linked kinases (ILKs) (Acconcia et al., 2007) which, in turn, phosphorylates the NICD at Ser2173 and promotes proteosome-mediated NICD degradation, a process in which the E3 ligase F-box/WD repeat-containing protein 7 (Fbxw7) [which tags NICD with ubiquitin (Ub)] plays an essential role (Mo et al., 2007). ILK, which plays an essential role in tumorigenesis, has been shown to regulate OPC proliferation and differentiation (Hussain & Macklin, 2017). In addition to ILK, NICD is also reported to interact directly with PAK1 and facilitate PAK1 (and/or ILK) nuclear translocation (Yoon et al., 2016). HDAC, histone deacetylase; NCoR, nuclear receptor co-repressor 2; RBP-J, recombination signal binding protein for immunoglobulin kappa J region.
Fig. 6.
Fig. 6.
Expression of group I p21-activated kinases (PAKs) in different types of white matter plaques and control white matter of the multiple sclerosis (MS) brain. Different types of MS plaques were isolated by laser capture microdissection and the resulting protein extract was subjected to proteomic identification (Han et al., 2008). Active plaques (AP) are histologically characterized by indistinct margins of demyelination and dense infiltration of myelin debris-loaded macrophages/microglia. Chronic active plaques (CAP), also known as smouldering plaques or slowly expanding plaques (Frischer et al., 2015) are characterized by defined demarcation of demyelination with no or few activated macrophages/microglia within the plaque but with many surrounding plaques. Chronic plaques (CP, or chronic inactive plaques) are characterized by a sharply demarcated demyelination edge with no or few myelin debris-loaded activated macrophages/microglia. ctrl, control. Data adapted from Supplemental Table 3 of Han et al. (2008).
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