EVOLUTIONARY HOMOLOGS part 3/3 || part 1/3 || part 2/3

PAKs and the cell cycle in yeast

The activity of the Saccharomyces cerevisiae pheromone signal transduction pathway is regulated by the cyclin/cdk dimer (Cln1/2-Cdc28 cyclin-dependent kinase). High level expression of CLN2 can repress activation of the pathway by mating factor. CLN2 overexpression can also repress FUS1 induction (FUS1 is a member of a family of proteins involved in cytokinesis or other actin-mediated processes) if the signaling pathway is activated at the level of the beta-subunit of a G-protein (STE4) but not when activated at the level of downstream kinases (STE20 and STE11) or at the level of the transcription factor STE12. This epistatic analysis indicates that repression of the pheromone signaling pathway by Cln2-Cdc28 kinase takes place at a level around STE20. In agreement with this, a marked reduction in the electrophoretic mobility of the Ste20 protein is observed at the time in the cell cycle of maximal expression of CLN2. This mobility change is constitutive in cells overexpressing CLN2 and absent in cells lacking CLN1 and CLN2. These changes in electrophoretic mobility correlate with repression of pheromone signaling and suggest Ste20 as a target for repression of signaling by G1 cyclins. Two morphogenic pathways for which Ste20 is essential, pseudohyphal differentiation and haploid-invasive growth, also require CLN1 and CLN2. Together with the previous observation that Cln1 and Cln2 are required for the function of Ste20 in cytokinesis, this suggests that Cln1 and Cln2 regulate the biological activity of Ste20 by promoting morphogenic functions, while inhibiting the mating factor signal transduction function (Oehlen, 1998).

Cyclins and cyclin-dependent kinases induce and coordinate the events of the cell cycle, although the mechanisms by which they do so remain largely unknown. In budding yeast, a pathway used by the Clb2 cyclin to control bud growth during mitosis provides a good model system in which to understand how cyclin-dependent kinases control cell-cycle events. In this pathway, Clb2 initiates a series of events that lead to the mitosis-specific activation of the Gin4 protein kinase. A protein called Nap1 is required in vivo for the activation of Gin4, and is able to bind to both Gin4 and Clb2. A simple genetic screen was used to identify additional proteins that function in this pathway. The Cdc42 GTPase and a member of the PAK kinase family called Cla4 both function in the pathway used by Clb2 to control bud growth during mitosis. Cdc42 and Cla4 interact genetically with Gin4 and Nap1, and both are required in vivo for the mitosis-specific activation of the Gin4 kinase. Furthermore, Cla4 undergoes a dramatic hyperphosphorylation in response to the combined activity of Nap1, the Clb2-Cdc28 kinase complex, and the GTP-bound form of Cdc42. Evidence is presented that suggests that the hyperphosphorylated form of Cla4 is responsible for relaying the signal to activate Gin4. Previous studies have suggested that cyclin-dependent kinases control the cell cycle by directly phosphorylating proteins involved in specific events, such as nuclear lamins, microtubule-associated proteins and histones. In contrast, these results demonstrate that the Clb2-Cdc28 cyclin-dependent kinase complex controls specific cell-cycle events through a pathway that involves a GTPase and at least two different kinases. This suggests that cyclin-dependent kinases may control many cell-cycle events through GTPase-linked signaling pathways that resemble the intricate signaling pathways known to control many other cellular events (Tjandra, 1998).

PAKs and the cell cycle in vertebrates

New members (X-PAKs) of the Ste20/PAK family of protein kinases have been identified in Xenopus, and their role in the process that maintains oocytes arrested in the cell cycle has been investigated. Microinjection of a catalytically inactive mutant of X-PAK1 with a K/R substitution in the ATP binding site, also deleted of its N terminal-half (which contains the conserved domains responsible for binding of both Cdc42/Rac GTPases and SH3-containing proteins), greatly facilitates oocyte release from G2/prophase arrest by progesterone and insulin. Addition of the same X-PAK1 mutant to cell cycle extracts from unfertilized eggs inducesapoptosis, as shown by activation of caspases and cytological changes in in vitro-assembled nuclei. This is suppressed by adding Bcl-2 or the DEVD peptide inhibitor of caspases, and is rescued by competing the dominant-negative mutant with its constitutively active X-PAK1 counterpart. Such results indicate that X-PAK1 (or another member of the Xenopus Ste20/PAK family of protein kinases) is involved in the arrest of oocytes at G2/prophase and prevention of apoptosis; thus death by apoptosis and release of healthy oocytes from cell cycle arrest may be linked. That cell cycle arrest protects oocytes from apoptosis is consistent with the finding that extracts from metaphase II-arrested oocytes are less sensitive to apoptotic signals than those from activated eggs (Faure, 1997).

X-PAKs are involved in negative control of the process of oocyte maturation in Xenopus. In the present study, the events targeted by the kinase in the inhibition of the G2/M transition have been defined more precisely. A constitutive truncated version of X-PAK1 (X-PAK1-Cter) does not prevent the association of cyclin B with p34(cdc2) but rather prevents the activation of the inactive complexes present in the oocyte. Microinjection of recombinant X-PAK1-Cter active kinase into progesterone-treated oocytes prevents c-Mos accumulation and activation of both MAPK and maturation-promoting factor (MPF). In conditions permissive for MAPK activation, MPF activation still fails. Proteins participating in the MPF amplification loop, including the Cdc25-activating Polo-like kinase are all blocked. Indeed, using active MPF, the amplification loop is not turned on in the presence of X-PAK1. These results indicate that X-PAK and protein kinase A targets in the control of oocyte maturation are similar, and furthermore that this negative regulation is not restricted to meiosis, because G2/M progression is also prevented in Xenopus cycling extracts in the presence of active X-PAK1 (Faure, 1999).

Mammalian p21-activated kinase 1 (Pak1) is a highly conserved effector for the small GTPases Cdc42 and Rac1. In lower eukaryotes, Pak1 homologs are regulated during the cell cycle by phosphorylation. Pak1 is phosphorylated during mitosis in mammalian fibroblasts. This phosphorylation occurs at a single site, Thr 212, within a domain that is unique to Pak1. Cdc2 phosphorylates Pak1 at the identical site in vitro, and inhibition of Cdc2 abolishes Pak1 mitotic phosphorylation in vivo, indicating that Cdc2 is the kinase responsible for phosphorylating Pak1 in mitotic cells. Expression of a Pak1 mutant in which Thr 212 is replaced with a phosphomimic (aspartic acid) has marked effects on the rate and extent of postmitotic spreading of fibroblasts. The mitotic phosphorylation of Pak1 does not alter the basal or Rac-stimulated activity of this kinase, but it does affect the coimmunoprecipitation of at least three proteins with Pak1. These findings are the first to implicate a mammalian Pak in cell cycle regulation and suggest that Pakl, as a result of phosphorylation by Cdc2, alters its association with binding partners and/or substrates that are relevant to the morphologic changes associated with cell division (Thiel, 2002).

The Pak kinases are targets of the Rho GTPases Rac and Cdc42, which regulate cell shape and motility. It is increasingly apparent that part of this function is due to the effect Pak kinases have on microtubule organization and dynamics. Overexpression of Xenopus Pak5 enhances microtubule stabilization, and Pak1 may inhibit a microtubule-destabilizing protein, Op18/Stathmin. A specific phosphorylation site has been identified on mammalian Pak1, T212, which is targeted by the neuronal p35/Cdk5 kinase. Pak1 phosphorylated on T212, Pak1T212(PO4), is enriched in axonal growth cones and colocalizes with small peripheral bundles of microtubules. Cortical neurons overexpressing a Pak1A212 mutant display a tangled neurite morphology, which suggests that the microtubule cytoskeleton is affected. Cyclin B1/Cdc2 phosphorylates Pak1 in cells undergoing mitosis. In the developing cortex and in cultured fibroblasts, Pak1T212(PO4) is enriched in microtubule-organizing centers and along parts of the spindles. In living cells, a peptide mimicking phosphorylated T212 accumulates at the centrosomes and spindles and causes an increased length of astral microtubules during metaphase or following nocodazole washout. It is proposed that the region surrounding phosphorylated T212 contains a protein binding site, since the phosphorylated peptide is enriched in spindles and MTOCs and competes with endogenous Pak1 for this location.Together these results suggest that similar signaling pathways regulate microtubule dynamics in a remodeling axonal growth cone and during cell division (Banerjee, 2002).

The Xenopus p21-activated kinase 3 (XPak3) has been isolated by virtue of its expression in the territory of primary neurogenesis in the developing embryo. XPak3, but not the other Pak variants, responds positively to X-Ngnr-1 and negatively to X-Notch-1. A constitutively active form of XPak3, generated by fusing a myristylation signal to the N-terminus (XPak3-myr), induces early cell cycle arrest at high concentrations, while ectopic expression of low amounts induces premature neuronal differentiation. Conversely, XPak3 loss of function achieved by use of an antisense morpholino oligonucleotide increases cell proliferation and inhibits neuronal differentiation; this phenotype is rescued by co-injection of XPak3-myr. It is concluded that XPak3 is a novel member of the proneural pathway, functioning downstream of neurogenin to withdraw neuronally programmed cells from the mitotic cell cycle, thus allowing for their differentiation (Souopgui, 2002).

Similar to what is reported for XPak3 with respect to its function in the context of cell cycle regulation, the two other, closely related but differentially expressed Pak variants in Xenopus, XPak1 and XPak2, have been reported to be involved in cell cycle control, negatively regulating the G2/M phase transition during the process of oocyte maturation. Interestingly, and similar to the results observed upon misexpression of an activated form of XPak3, microinjection of a truncated, catalytically active form of Pak1 was reported to induce cleavage arrest in early Xenopus embryos. Furthermore, it has been demonstrated that a constitutively active deletion mutant derived from XPak1 blocks progesterone-induced oocyte maturation. A negative regulatory function in meiotic cell cycle progression has also been reported for XPak2. Phosphorylation of XPak2, which may be directly linked to maturation-promoting factor (MPF) activity, results in its inactivation, allowing for maturation to proceed to completion. At present, it is not known which partner molecules would be relevant for the cell cycle regulatory activities that are describe for XPak3 in the context of neuronal differentiation, but the more common functional link of different Pak variants to cell cycle control indicates that these different Pak proteins may be elements of similarly organized functional networks. Interestingly, it has been found that the Drosophila Pak is required for photoreceptor axon guidance. In the development stages following primary neurogenesis, XPak3 is strongly expressed throughout the central nervous system and in the developing eye; in the late stages, XPak3 may serve additional functions, perhaps including axonal guidance (Souopgui, 2002).

Cell cycle regulatory activities in the context of different aspects of neural development in Xenopus have been ascribed to p27XIC1, a Cdk inhibitor. During earlier phases of development, regulation of p27XIC1 expression may depend on XBF-1, a winged-helix transcription factor that is expressed specifically in the anterior neural plate. Similar to the effects observed upon inactivation of XPak3, high concentrations of XBF-1 result in suppression of neuronal differentiation and expansion of the undifferentiated neuroectoderm, as well as in a direct suppression of p27XIC1 gene transcription. However, the pattern of expression for these genes does not match that characteristic for N-tubulin, i.e. primary neurogenesis (Souopgui, 2002).

PAK and gastrulation

The p21-activated kinase (PAK) proteins regulate many cellular events including cell cycle progression, cell death and survival, and cytoskeleton rearrangements. X-PAK5 binds the actin and microtubule networks, and can potentially regulate their coordinated dynamics during cell motility. The functional importance of this kinase during gastrulation was examined in Xenopus. X-PAK5 is mainly expressed in regions of the embryo that undergo extensive cell movements during gastrula such as the animal hemisphere and the marginal zone. Expression of a kinase-dead mutant inhibits convergent extension movements in whole embryos and in activin-treated animal cap by modifying behavior of cells. This phenotype is rescued in embryo by adding back X-PAK5 catalytic activity. The active kinase decreases cell adhesiveness when expressed in animal hemisphere and inhibits the calcium-dependent reassociation of cells, while dead X-PAK5 kinase localizes to cell-cell junctions and increases cell adhesion. In addition, endogenous X-PAK5 colocalizes with adherens junction proteins and its activity is regulated by extracellular calcium. Taken together, these results suggest that X-PAK5 regulates convergent extension movements in vivo by modulating the calcium-mediated cell-cell adhesion (Faure, 2005).

Serotonin-induced regulation of the actin network for learning-related synaptic growth requires Cdc42, N-WASP, and PAK in Aplysia sensory neurons

Application of Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, at the Aplysia sensory to motor neuron synapse blocks long-term facilitation (LTF) and the associated growth of new sensory neuron varicosities induced by repeated pulses of serotonin (5-HT). cDNAs encoding Aplysia Rho, Rac, and Cdc42 have been isolated and it has been found that Rho and Rac had no effect but that overexpression in sensory neurons of a dominant-negative mutant of ApCdc42 or the CRIB domains of its downstream effectors PAK and N-WASP selectively reduces the long-term changes in synaptic strength and structure. FRET analysis indicates that 5-HT activates ApCdc42 in a subset of varicosities contacting the postsynaptic motor neuron and that this activation is dependent on the PI3K and PLC signaling pathways. The 5-HT-induced activation of ApCdc42 initiates reorganization of the presynaptic actin network leading to the outgrowth of filopodia, some of which are morphological precursors for the learning-related formation of new sensory neuron varicosities (Udo, 2005).

Actin is enriched in both the pre- and postsynaptic compartments of most neurons. Although the activity-dependent modulation of actin dynamics at postsynaptic spines has been well documented, the extent and role of actin regulation in presynaptic terminals is not well understood. During development, reorganization of actin in growth cones has been shown to play an important role in axonal pathfinding. However, in mature neurons, it has been suggested that the presynaptic actin network functions more as an intracellular scaffold rather than as a propulsive force, that contributes directly to the type of frank structural remodeling reported for postsynaptic dendritic spines. In Aplysia, repeated applications of 5-HT (which lead to LTF) induce a slower and more persistent alteration in the dynamics of the presynaptic actin network, leading to the growth of new sensory neuron synapses. The data indicate that Cdc42 is one of the key molecular regulators of this learning-related modulation of presynaptic actin organization (Udo, 2005).

The family of Rho GTPases has been shown to play an important role in neuronal development, for example, the establishment of polarity, axon guidance, and the growth and maintenance of dendritic spines. ApCdc42 is involved in long-term synaptic plasticity in Aplysia, suggesting that Cdc42 may also have a role in learning and memory storage in the mature nervous system. It was surprising that Rac, which is functionally related to Cdc42 and known to regulate spine morphology and memory consolidation in mice, does not significantly contribute to LTF and the associated structural changes. By contrast, Rho tends to oppose the effects of Cdc42 on long-term synaptic plasticity, which is consistent with the ways in which these two proteins regulate actin dynamics (Udo, 2005).

In Aplysia, the activation of ApCdc42 in sensory neurons leads to the outgrowth of filopodia from presynaptic varicosities. Interestingly, 5-HT stimulation by itself naturally induces filopodia; this induction is dependent on the activation of ApCdc42. Filopodia have been proposed to be morphological precursors of dendritic spines in the mammalian central nervous system, and this process may be regulated by neuronal activity. The 5-HT-induced activation of Cdc42 in Aplysia triggers not only the formation of filopodia but also the molecular maturation of neurotransmitter release sites. A major synaptic vesicle protein, synaptophysin, accumulates at the tips of 5-HT-induced filopodia, some of which then give rise to new varicosities. These observations support the following ideas: (1) filopodia represent one type of morphological precursor for the growth of new presynaptic varicosities during learning-related synaptic plasticity, and (2) the formation of filopodia and initial assembly of the presynaptic compartment can be induced by the activation of Cdc42 (Udo, 2005).

5-HT is a modulatory neurotransmitter released from facilitating interneurons that make synaptic contacts onto sensory neurons. Most of the 5-HT receptors are known to be G protein coupled, and G proteins such as Gα12 and Gα13 have been shown to link to Rho. However, the signaling pathways for Rac/Cdc42 appear to be different from those for Rho, and the molecular cascade between G protein-coupled receptors and Cdc42 is not well understood. The current results suggest that 5-HT activates ApCdc42 through pathways involving PLC and PI3 kinase. PLC produces two independent second messengers (diacylglycerol and inositol triphosphate) to initiate a variety of cellular functions. It has also been shown that some isoforms of PLC are able to directly bind to Rac/Cdc42 and to enhance its activity. Since an increase in the internal concentration of calcium is known to stimulate actin polymerization, PLC may send multiple signals to regulate actin-related structures. Like PLC, PI3 kinase also plays a key role in regulating cell growth and survival. PI3 kinase is thought to interact directly with Cdc42 and stimulate its activity. Although PI3 kinase is usually activated by receptor tyrosine kinases such as Trk receptors, recent evidence suggests that some PI3 kinase isoforms (such as type 1B) can be upregulated by their interaction with Gβγ. Thus, it is possible that PI3 kinase may be activated by 5-HT receptors as well as Trk-like receptors and that PLC and PI3K may send signals to ApCdc42 via independent pathways (Udo, 2005).

The synapse-specific nature of the 5-HT-induced activation of ApCdc42 suggests the possibility of a coordinated interaction between the presynaptic sensory neuron and the postsynaptic motor neuron. This could be mediated by a variety of different cell adhesion or trans-synaptic signaling molecules. For example, ephexin and IQGAP, which bind to the EphA receptor and cadherin, respectively, are known to modulate the activity of Cdc42. The identification of such molecules in Aplysia should provide additional molecular insights into the upstream signaling pathways that activate ApCdc42 (Udo, 2005).

The differential activation of ApCdc42 at a spatially distinct subset of presynaptic sensory neuron varicosities is consistent with previous studies, which have shown that the initial segment and cell body of the postsynaptic motor neuron is a preferred site for new sensory neuron varicosity formation induced by 5-HT. It is proposed that 5-HT receptors coupled to G proteins (Gαq and Gβγ) activate the PLC and PI3 kinase pathways, which in turn upregulate ApCdc42 at specific presynaptic varicosities. The selective activation of ApCdc42 leads to the formation of actin-based filopodia by activating downstream effector proteins such as N-WASP and to a lesser extent PAK. Presynaptic components, including synaptic vesicles, appear to be recruited to the tips of specific filopodia, possibly through actin-myosin-dependent transport, which then become transformed into new functional sensory neuron varicosities. Thus, 5-HT-induced regulation of the Cdc42 signaling pathways and the consequent reorganization of the presynaptic actin network appear to be a part of the initial molecular cascade required for the growth of new sensory neuron synapses associated with long-term memory (Udo, 2005).

Essential role for the Pak4 protein kinase in extraembryonic tissue development and vessel formation

Pak4 is a member of the group B family of Pak serine/threonine kinases, originally identified as an effector protein for the Rho GTPase Cdc42. Pak4 knockout mice are embryonic lethal and do not survive past embryonic day 11.5. Previous work on Pak4 knockout mice has focused on studying the phenotype of the embryo. Abnormalities in the extraembryonic tissue, however, are common causes of early embryonic death in knockout mice. Extraembryonic tissue associated with the Pak4-null embryos was therefore examined. Abnormalities in both yolk sacs and placentas resulted when Pak4 was deleted. These included a lack of vasculature throughout the extraembryonic tissue, as well as an abnormally formed labyrinthine layer of the placenta. Interestingly, epiblast-specific deletion of Pak4 using a conditional knockout system, did not rescue the embryonic lethality. In fact, it did not even rescue the extraembryonic tissue defects. These results suggest that the extraembryonic tissue abnormalities are secondary to defects that occur in response to epiblast abnormalities. More detailed analysis suggests that abnormalities in vasculature throughout the extraembryonic tissue and the epiblast may contribute to the death of the Pak4-null embryos (Tian, 2009).

A key role for Pak4 in proliferation and differentiation of neural progenitor cells

The Pak4 serine/threonine kinase regulates cytoskeletal organization, and controls cell growth, proliferation, and survival. Deletion of Pak4 in mice results in embryonic lethality prior to embryonic day 11.5. Pak4 knockout embryos exhibit abnormalities in the nervous system, the heart, and other tissues. In this study a conditional deletion of Pak4 was generated in order to study the function of Pak4 in the development of the brain. Nervous system-specific conditional deletion of Pak4 was accomplished by crossing mice with a floxed allele of Pak4 with transgenic mice expressing Cre recombinase under the control of the nestin promoter. The conditional Pak4 knockout mice were born normally, but displayed growth retardation and died prematurely. The brains showed a dramatic decrease in proliferation of cortical and striatal neuronal progenitor cells. In vitro analyses revealed a reduced proliferation and self-renewing capacity of neural progenitor cells isolated from Pak4 knockout brains. The mice also exhibited cortical thinning, impaired neurogenesis and loss of neuroepithelial adherens junctions. By the time the mice died, by 4 weeks after birth, severe hydrocephalus could also be seen. These results suggest that Pak4 plays a critical role in the regulation of neural progenitor cell proliferation and in establishing the foundation for development of the adult brain (Tian, 2011).

Agrin as a Mechanotransduction Signal Regulating YAP through the Hippo Pathway

The Hippo pathway effectors YAP and TAZ (see Drosophila Yorkie) act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM) cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. This study uncovered a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 (see Drosophila Pak) signaling and negating the functions of Merlin and LATS1/2 (see Drosophila Merlin and Warts). It was further shown that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. It is proposed that Agrin acts as a mechanotransduction signal in the ECM (Chakraborty, 2017).

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PAK-kinase: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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