Mixed synthetic oligonucleotides encoding a sequence conserved among tyrosine-specific protein kinases were used to probe the genome of the fission yeast Schizosaccharomyces pombe. A single gene (kin1+) was isolated that encodes a putative protein kinase closely related to the KIN1- and KIN2-encoded serine/threonine-specific protein kinases of Saccharomyces cerevisiae. kin1+ is transcribed into a 3.5-kilobase mRNA that contains an uninterrupted open reading frame encoding a polypeptide of 98 kDa. In contrast to results obtained with kin mutants of S. cerevisiae, disruption of the Sc. pombe kin1+ gene results in recessive morphological and growth defects. kin1-disrupted cells grow slowly on enriched medium and grow as spheres, in contrast to wild-type Sc. pombe cells, which grow as rods. Relative to kin1+ cells, kin1-disrupted cells are differentially sensitive to lysis by treatment with alpha- and beta-glucanases, suggesting an alteration in either the composition or the organization of their cell walls (Levin, 1990).
The kin1 protein kinase of the fission yeast Schizosaccharomyces pombe is a member of the PAR-1/MARK (partitioning-defective 1/microtubule-associated protein/microtubule affinity-regulating kinase) family important in eukaryotic cell polarity and cytoskeletal dynamics. kin1 plays a role in establishing the characteristic rod-shaped morphology of fission yeast. Cells in which kin1 was deleted are viable but are impaired in growth, and are rounded at one end or both ends. They are monopolar because after mitosis they fail to activate bipolar growth, and are delayed in cytokinesis, resulting in a high proportion of septated cells often with multiple septa. This phenotype can be partially rescued by heterologous expression of human MARK proteins, which restore bipolar growth in most cells, but do not correct the delay in cytokinesis. Using chromosomal epitope tagging, it has been shown that kin1p localizes to the cell ends, except during mitosis when it disappears from cell ends. After mitosis, kin1p first reappears at the new cell end. Overexpression of kin1 results in a loss of polarity, with partially or fully rounded cells. From these results it is suggested that kin1 is required to direct the growth machinery to the cell ends (Drewes, 2003).
Eight strict maternal effect mutations identifying four genes, par-1, par-2, par-3, and par-4, required for cytoplasmic localization in early embryos of the nematode C. elegans have been isolated and analyzed. Mutations in these genes lead to defects in cleavage patterns, timing of cleavages, and localization of germ line-specific P granules. Four mutations in par-1 and par-4 are fully expressed maternal effect lethal mutations; all embryos from mothers homozygous for these mutations arrest as amorphous masses of differentiated cells but are specifically lacking intestinal cells. Four mutations in par-2, par-3, and par-4 are incompletely expressed maternal effect lethal mutations and are also grandchildless; some embryos from homozygous mothers survive and grow to become infertile adults due to the absence of functional germ cells. It is proposed that all of these defects result from the failure of a maternally encoded system for intracellular localization in early embryos (Kemphues, 1988).
The autonomous or cell-intrinsic developmental properties of early embryonic blastomeres in nematodes are thought to result from the action of maternally provided determinants. After the first cleavage of the C. elegans embryo, only the posterior blastomere, P1, has a cell-intrinsic ability to produce pharyngeal cells. The product of the maternal gene skn-1 is required for P1 to produce pharyngeal cells. The Skn-1 protein is nuclear localized and P1 appears to accumulate markedly higher levels of Skn-1 protein than its sister, the AB blastomere. The distribution of Skn-1 protein was examined in embryos from mothers with maternal-effect mutations in the genes mex-1, par-1, and pie-1. These results suggest that mex-1(+) and par-1(+) activities are required for the unequal distribution of the Skn-1 protein and that pie-1(+) activity may function to regulate the activity of Skn-1 protein in the descendants of the posterior blastomere P1 (Bowerman, 1993).
The first cleavage of C. elegans is asymmetric, generating daughter cells with different sizes, cytoplasmic components, and fates. Mutations in the par-1 gene disrupt this asymmetry. par-1 encodes a putative Ser/Thr kinase with similarity to kinases from yeasts and mammals. Two strong alleles have mutations in the kinase domain, suggesting that kinase activity is essential for par-1 function. PAR-1 protein is localized to the posterior periphery of the zygote and is distributed in a polar fashion preceding the asymmetric divisions of the germline lineage. Because PAR-1 distribution in the germline correlates with the distribution of germline-specific P granules, it is possible that PAR-1 functions in germline development as well as in establishing embryonic polarity (Guo, 1995).
The par-3 gene is required for establishing polarity in early C. elegans embryos. Embryos from par-3 homozygous mothers show defects in segregation of cytoplasmic determinants and in positioning of the early cleavage spindles. The PAR-3 protein is asymmetrically distributed at the periphery of the zygote and asymmetrically dividing blastomeres of the germline lineage. The PAR-3 distribution is roughly the reciprocal of PAR-1, another protein required for establishing embryonic polarity in C. elegans. Analysis of the distribution of PAR-3 and PAR-1 in other par mutants reveals that par-2 activity is required for proper localization of PAR-3 and that PAR-3 is required for proper localization of PAR-1. In addition, the distribution of the PAR-3 protein correlates with differences in cleavage spindle orientation and suggests a mechanism by which PAR-3 contributes to control of cleavage pattern (Etemad-Moghadam, 1995).
Daughter cells with distinct fates can arise through intrinsically asymmetrical divisions. Before such divisions, factors crucial for determining cell fates become asymmetrically localized in the mother cell. In Caenorhabditis elegans, PAR proteins are required for the early asymmetrical divisions that establish embryonic polarity, and are asymmetrically localized in early blastomeres, although the mechanism of their distribution is not known. Nonmuscle myosin II heavy chain (designated NMY-2) has been identified in C. elegans by means of its interaction with the PAR-1 protein, a putative Ser/Thr protein kinase. Injections of nmy-2 antisense RNA into ovaries of adult worms causes embryonic partitioning defects and leads to mislocalization of PAR proteins. It is therefore concluded that the NMY-2 is required for establishing cellular polarity in C. elegans embryos (Guo, 1996).
The par genes participate in the process of establishing cellular asymmetries during the first cell cycle of Caenorhabditis elegans development. The par-2 gene is required for the unequal first cleavage and for asymmetries in cell cycle length and spindle orientation in the two resulting daughter cells. The PAR-2 protein is present in adult gonads and early embryos. In gonads, the protein is uniformly distributed at the cell cortex, and this subcellular localization depends on microfilaments. In the one-cell embryo, PAR-2 is localized to the posterior cortex and is partitioned into the posterior daughter, P1, at the first cleavage. PAR-2 exhibits a similar asymmetric cortical localization in P1, P2, and P3, the asymmetrically dividing blastomeres of germ line lineage. This distribution in embryos is very similar to that of PAR-1 protein. By analyzing the distribution of the PAR-2 protein in various par mutant backgrounds, proper asymmetric distribution of PAR-2 depends on par-3 activity but not upon par-1 or par-4. par-2 activity is required for proper cortical localization of PAR-1 and this effect requires wild-type par-3 gene activity. Although par-2 activity is not required for posterior localization of P granules at the one-cell stage, it is required for proper cortical association of P granules in P1 (Boyd, 1996).
After fertilization in C. elegans, activities encoded by the maternally expressed par genes appear to establish cellular and embryonic polarity. Loss-of-function mutations in the par genes disrupt A/P asymmetries in early embryos and result in highly abnormal patterns of cell fate. Little is known about how the early asymmetry defects are related to the cell fate patterning defects in par mutant embryos, or about how the par gene products affect the localization and activities of developmental regulators known to specify the cell fate patterns made by individual blastomeres. Examples of such regulators of blastomere identity include the maternal proteins MEX-3 and GLP-1, expressed at high levels anteriorly, and SKN-1 and PAL-1, expressed at high levels posteriorly in early embryos. To better define par gene functions, the expression patterns of MEX-3, PAL-1 and SKN-1 were examined, and mex-3, pal-1, skn-1 and glp-1 activities were examined in par mutant embryos. Mutational inactivation of each par gene results in a unique phenotype, but in no case is a complete loss of A/P asymmetry observed. It is concluded that no one par gene is required for all A/P asymmetry and it is suggested that, in some cases, the par genes act independent of one another to control cell fate patterning and polarity. Discussed are the implications of these findings for the furtherance of an understanding of how the initial establishment of polarity in the zygote by the par gene products leads to the proper localization of more specifically acting regulators of blastomere identity (Bowerman, 1997).
The translation of maternal glp-1 mRNA is regulated both temporally and spatially in the early C. elegans embryo. To investigate the control of embryonic glp-1 expression, the distribution of GLP-1 protein was examined in selected maternal effect mutants that affect pattern or fate in the early embryo. Mutants that disrupt anterior-posterior asymmetry in the early embryo [par-1-par-6, emb-8, Par(q537)] disrupt the spatial but not temporal control of GLP-1 expression: GLP-1 is observed at the normal stage of embryogenesis in par-like mutants; however, it is uniformly distributed. In contrast, mutants that alter blastomere identity (skn-1, pie-1, mex-1, apx-1) do not affect the normal GLP-1 pattern. It is concluded that genes controlling the asymmetry of cellular components, including P granules, also control GLP-1 asymmetry in the early embryo. The finding that mutants that disrupt anterior-posterior asymmetry translate GLP-1 in all blastomeres suggests that loss of embryonic asymmetry causes translational activation of GLP-1 in the posterior (Crittenden, 1997).
Establishment of A/P polarity in the C. elegans embryo depends on filamentous (F-) actin. An F-actin-binding protein that is enriched in the anterior cortex of the one-cell embryo is hypothesized to link developmental polarity to the actin cytoskeleton. This protein, POD-1, as a new member of the coronin family of actin-binding proteins. A deletion has been genereated within the pod-1 gene. Elimination of POD-1 from early embryos results in a loss of physical and molecular asymmetries along the A/P axis. For example, PAR-1 and PAR-3, which themselves are polarized and required for A/P polarity, are delocalized in pod-1 mutant embryos. However, unlike loss of PAR proteins, loss of POD-1 gives rise to the formation of abnormal cellular structures, namely large vesicles of endocytic origin, membrane protrusions, unstable cell divisions, a defective eggshell, and deposition of extracellular material. It is concluded that, analogous to coronin, POD-1 plays an important role in intracellular trafficking and organizing specific aspects of the actin cytoskeleton. Models have been put forth, attempting to explain how the role of POD-1 in basic cellular processes could be linked to the generation of polarity along the embryonic A/P axis (Rappleye, 1999).
The KH domain protein MEX-3 is central to the temporal and spatial control of PAL-1 expression in the C. elegans early embryo. PAL-1 is a Caudal-like homeodomain protein that is required to specify the fate of posterior blastomeres. While pal-1 mRNA is present throughout the oocyte and early embryo, PAL-1 protein is expressed only in posterior blastomeres, starting at the four-cell stage. To better understand how PAL-1 expression is regulated temporally and spatially, MEX-3 interacting proteins (MIPs) have been identified and two that are required for the patterning of PAL-1 expression are described in detail. RNA interference of MEX-6, a CCCH zinc-finger protein, or SPN-4, an RNA recognition motif protein, causes PAL-1 to be expressed in all four blastomeres starting at the four-cell stage. Genetic analysis of the interactions between these mip genes and the par genes, which provide polarity information in the early embryo, defines convergent genetic pathways that regulate MEX-3 stability and activity to control the spatial pattern of PAL-1 expression. These experiments suggest that par-1 and par-4 affect distinct processes. par-1 is required for many aspects of embryonic polarity, including the restriction of MEX-3 and MEX-6 activity to the anterior blastomeres. PAL-1 is not expressed in par-1 mutants, because MEX-3 and MEX-6 remain active in the posterior blastomeres. The role of par-4 is less well understood. This analysis suggests that par-4 is required to inactivate MEX-3 at the four-cell stage. Thus, PAL-1 is not expressed in par-4 mutants because MEX-3 remains active in all blastomeres. It is proposed that MEX-6 and SPN-4 act with MEX-3 to translate the temporal and spatial information provided by the early acting par genes into the asymmetric expression of the cell fate determinant PAL-1 (Huang, 2002).
Polarization of the C. elegans zygote along the anterior-posterior axis depends on cortically enriched (PAR) and cytoplasmic (MEX-5/6) proteins, which function together to localize determinants (e.g. PIE-1) in response to a polarizing cue associated with the sperm asters. Using time-lapse microscopy and GFP fusions, the localization dynamics of PAR-2, PAR-6, MEX-5, MEX-6 and PIE-1 were studied in wild-type and mutant embryos. These studies reveal that polarization involves two genetically and temporally distinct phases. During the first phase (establishment), the sperm asters at one end of the embryo exclude the PAR-3/PAR-6/PKC3 complex from the nearby cortex, allowing the ring finger protein PAR-2 to accumulate in an expanding `posterior' domain. Onset of the establishment phase involves the non-muscle myosin NMY-2 and the 14-3-3 protein PAR-5. The kinase PAR-1 and the CCCH finger proteins MEX-5 and MEX-6 also function during the establishment phase in a feedback loop to regulate growth of the posterior domain. The second phase begins after pronuclear meeting, when the sperm asters begin to invade the anterior. During this phase (maintenance), PAR-2 maintains anterior-posterior polarity by excluding the PAR-3/PAR-6/PKC3 complex from the posterior. These findings provide a model for how PAR and MEX proteins convert a transient asymmetry into a stably polarized axis (Cuenca, 2003).
The establishment phase requires the class II non-muscle myosin, NMY-2: nmy-2(RNAi) prevents PAR-6 (and presumably associated PAR-3 and PKC-3) from sensing the polarity cue, causing it to remain uniformly distributed throughout the cortex. In NMY-2-depleted embryos, PAR-2 is prevented from accumulating at the cortex by PAR-6 (and/or its partners). This 'default' state of PAR-6 on/PAR-2 off is also observed in mutants lacking sperm asters and in mutants where the MTOC detaches from the cortex prematurely. These observations suggest that the initial symmetry-breaking event involves signaling between the MTOC and the actin cytoskeleton. Consistent with this view, one of the earliest signs of polarization is cessation of ruffling in the cortex nearest the MTOC. Cessation of ruffling correlates with MTOC formation, but does not appear to require PAR activity (cessation of ruffling was observed in all par mutants examined in this study). These observations suggest that modification of the actin cytoskeleton may be an obligatory step before the onset of PAR asymmetry. It is proposed that signaling from the MTOC modifies the actin cytoskeleton locally, which causes the PAR-3/PAR-6/PKC-3 complex to become destabilized, allowing PAR-2 to accumulate in its place (Cuenca, 2003).
Surprisingly, it was found that the predominantly cytoplasmic MEX-5 and MEX-6 also play a role during the establishment phase. In the absence of MEX-5 and MEX-6, the posterior domain occasionally does not form (15%-30% of embryos), and frequently (50% or more of embryos) is slow to reach its final configuration. These observations indicate that, although MEX-5 and MEX-6 are not absolutely required for PAR localization in the zygote, they do play a role in ensuring a robust response by the PAR-3/PAR-6/PKC-3 complex to the MTOC/actin cytoskeleton signal (Cuenca, 2003).
This aspect of MEX-5/6 function is negatively regulated by PAR-1. In par-1 mutants, MEX-5 and MEX-6 cause the posterior domain to extend further towards the anterior during the establishment phase. Since PAR-1 itself becomes enriched in the posterior domain, one attractive possibility is that PAR-1 and MEX-5/6 participate in a feedback loop that limits expansion of the posterior domain. It is proposed that at the beginning of the establishment phase, MEX-5 and MEX-6 levels are high throughout the zygote and help clear the PAR-3/PAR-6/PKC-3 complex from the region nearest the sperm asters. This clearing allows PAR-2 and PAR-1 to accumulate on the cortex, which in turn reduces MEX-5/6 activity and/or levels in the surrounding cytoplasm. Eventually, MEX-5/6 levels become too low to fuel further expansion of the posterior domain. It is not yet known whether the partial penetrance of the mex-5(-);mex-6(-) phenotype is due to redundancy with other factors, or is indicative of a minor role for the feedback loop in regulating PAR asymmetry (Cuenca, 2003).
The existence of distinct establishment and maintenance phases is also supported by the observation that cdc-42 is required after prophase, but not earlier, for PAR-3, PAR-6 and PKC-3 asymmetry. Analysis of GFP:PAR-6 dynamics in par-1(RNAi) embryos suggests that PAR-1 also contributes to maintenance of PAR asymmetry after pronuclear meeting. How PAR-2, CDC-42 and PAR-1 function together to maintain the balance between anterior and posterior PAR domains remains to be determined (Cuenca, 2003).
The Caenorhabditis elegans vulva provides a simple model for the genetic analysis of pattern formation and organ morphogenesis during metazoan development. An essential role for the polarity protein PAR-1 in the development of the vulva has been discovered. Postembryonic RNA interference of PAR-1 causes a protruding vulva phenotype. Depleting PAR-1 during the development of the vulva has no detectable effect on fate specification or precursor proliferation, but instead seems to specifically alter morphogenesis. Using an apical junction-associated GFP marker, PAR-1 depletion was found to cause a failure of the two mirror-symmetric halves of the vulva to join into a single, coherent organ. The cells that normally form the ventral vulval rings fail to make contact or adhere and consequently form incomplete toroids, and dorsal rings adopt variably abnormal morphologies. PAR-1 undergoes a redistribution from apical junctions to basolateral domains during morphogenesis. Despite a known role for PAR-1 in cell polarity, no detectable differences has been observed in the distribution of various markers of epithelial cell polarity. It is proposed that PAR-1 activity at the cell cortex is critical for mediating cell shape changes, cell surface composition, or cell signaling during vulval morphogenesis (Hurd, 2003).
pEg3 is a Xenopus protein kinase related to members of the KIN1/PAR-1/MARK family. The founding members of this newly emerging kinase family are involved in the establishment of cell polarity and both microtubule dynamic and cytoskeleton organization. Sequence analyses suggest that pEg3 and related protein kinases in human, mouse, and Caenorhabditis elegans might constitute a distinct group in this family. pEg3 is encoded by a maternal mRNA, polyadenylated in unfertilized eggs and specifically deadenylated in embryos. In addition to an increase in expression, pEg3 is phosphorylated during oocyte maturation. Phosphorylation of pEg3 is cell cycle dependent during Xenopus early embryogenesis and in synchronized cultured XL2 cells. In embryos, the kinase activity of pEg3 is correlated to its phosphorylation state and is maximum during mitosis. Using Xenopus egg extracts it has been demonstrated that phosphorylation occurs at least in the noncatalytic domain of the kinase, suggesting that this domain might be important for pEg3 function (Blot, 2002).
Aberrant phosphorylation of the microtubule-associated protein tau is one of the pathological features of neuronal degeneration in Alzheimer's disease. The phosphorylation of Ser-262 within the microtubule binding region of tau is of particular interest because so far it is observed only in Alzheimer's disease and because phosphorylation of this site alone dramatically reduces the affinity for microtubules in vitro. A protein-serine kinase has been purified and characterized from brain tissue with an apparent molecular mass of 110 kDa on SDS gels. This kinase specifically phosphorylates tau on its KIGS or KCGS motifs in the repeat domain, whereas no significant phosphorylation outside this region was detected. Phosphorylation occurs mainly on Ser-262 located in the first repeat. This largely abolishes tau's binding to microtubules and makes the microtubules dynamically unstable, in contrast to other protein kinases that phosphorylate tau at or near the repeat domain. The data suggest a role for this novel kinase in cellular events involving rearrangement of the microtuble-associated proteins/microtubule arrays and their pathological degeneration in Alzheimer's disease (Drewes, 1995).
The phosphorylation of microtubule-associated proteins (MAPs) is thought to be a key factor in the regulation of microtubule stability. Recently, a novel protein kinase, termed p110 microtubule-affinity regulating kinase ('MARK'), has been shown to phosphorylate microtubule-associated protein tau at the KXGS motifs in the region of internal repeats and to cause the detachment of tau from microtubules. p110mark phosphorylates analogous KXGS sites in the microtubule binding domains of the neuronal MAP2 and the ubiquitous MAP4. Phosphorylation in vitro leads to the dissociation of MAP2 and MAP4 from microtubules and to a pronounced increase in dynamic instability. Thus, the phosphorylation of the repeated motifs in the microtubule binding domains of MAPs by p110mark might provide a mechanism for the regulation of microtubule dynamics in cells (Illenberger, 1996).
MARK phosphorylates the microtubule-associated proteins tau, MAP2, and MAP4 on their microtubule-binding domain, causing their dissociation from microtubules and increased microtubule dynamics. Describe here is the molecular cloning, distribution, activation mechanism, and overexpression of two MARK proteins from rat that arise from distinct genes. They encode Ser/Thr kinases of 88 and 81 kDa, respectively, and show similarity to the yeast kin1+ and C. elegans par-1 genes that are involved in the establishment of cell polarity. Expression of both isoforms is ubiquitous, and homologous genes are present in humans. Catalytic activity depends on phosphorylation of two residues in subdomain VIII. Overexpression of MARK in cells leads to hyperphosphorylation of MAPs on KXGS motifs and to disruption of the microtubule array, resulting in morphological changes and cell death (Drewes, 1997).
The establishment of polarity in the embryo is fundamental for the correct development of an organism. The first cleavage of the C. elegans embryo is asymmetric with certain cytoplasmic components being distributed unequally between the daughter cells. Six par genes (partition-defective) have been identified that are involved in the process of asymmetric division. One of these genes encodes a highly conserved protein, PAR-1, which is a serine/threonine kinase that localizes asymmetrically to the posterior part of the zygote and to those blastocysts that give rise to the germ line. It was reasoned that the mammalian homolog of PAR-1 (mPAR-1) might be involved in the process of polarization of epithelial cells, which consist of apical and basolateral membrane domains. mPAR-1 is expressed in a wide variety of epithelial tissues and cell lines and is associated with the cellular cortex. In polarized epithelial cells, mPAR-1 is asymmetrically localized to the lateral domain. A fusion protein lacking the kinase domain has the same localization as the full-length protein but its prolonged expression acts in a dominant-negative fashion: lateral adhesion of the transfected cells to neighboring cells is diminished, resulting in the former cells being 'squeezed out' from the monolayer. Moreover, the polarity of these cells is disturbed, resulting in mislocalization of E-cadherin. Thus, in the C. elegans embryo and in epithelial cells, polarity appears to be governed by similar mechanisms (Bohm, 1997).
Microtubules serve as transport tracks in molecular mechanisms governing cellular shape and polarity. Rapid transitions between stable and dynamic microtubules are regulated by several factors, including microtubule-associated proteins (MAPs). MAP/microtubule affinity regulating kinases (MARK) can phosphorylate the microtubule-associated-proteins MAP4, MAP2c, and tau on their microtubule-binding domain in vitro. This leads to their detachment from microtubules (MT) and an increased dynamic instability of MT. MARK protein kinases phosphorylate MAP2 and MAP4 on their microtubule-binding domain in transfected CHO cells. In CHO cells expressing MARK1 or MARK2 under control of an inducible promoter, MARK2 phosphorylates an endogenous MAP4-related protein. Prolonged expression of MARK2 results in microtubule-disruption, detachment of cells from the substratum, and cell death. Concomitant with microtubule disruption, a breakdown of the vimentin network is also observed, whereas actin fibers remain unaffected. Thus, MARK seems to play an important role in controlling cytoskeletal dynamics (Ebneth, 1999).
One of the hallmarks of Alzheimer's disease is the abnormal state of the microtubule-associated protein tau in neurons. It is both highly phosphorylated and aggregated into paired helical filaments, and it is commonly assumed that the hyperphosphorylation of tau causes its detachment from microtubules and promotes its assembly into PHFs. The relationship between the phosphorylation of tau by several kinases (MARK, PKA, MAPK, GSK3) and its assembly into PHFs has been examined. The proline-directed kinases MAPK and GSK3 are known to phosphorylate most Ser-Pro or Thr-Pro motifs in the regions flanking the repeat domain of tau: they induce the reaction with several antibodies diagnostic of Alzheimer PHFs, but this type of phosphorylation has only a weak effect on tau-microtubule interactions and on PHF assembly. By contrast, MARK and PKA phosphorylate several sites within the repeats (notably the KXGS motifs including Ser262, Ser324, and Ser356, plus Ser320); in addition PKA phosphorylates some sites in the flanking domains, notably Ser214. This type of phosphorylation strongly reduces tau's affinity for microtubules, and at the same time inhibits tau's assembly into PHFs. Thus, contrary to expectations, the phosphorylation that detaches tau from microtubules does not prime it for PHF assembly, but rather inhibits it. Likewise, although the phosphorylation sites on Ser-Pro or Thr-Pro motifs are the most prominent ones on Alzheimer PHFs (by antibody labeling), they are only weakly inhibitory to PHF assembly. This implies that the hyperphosphorylation of tau in Alzheimer's disease is not directly responsible for the pathological aggregation into PHFs; on the contrary, phosphorylation protects tau against aggregation (Schneider, 1999).
Tau is a microtubule-associated protein (MAP) that is functionally modulated by phosphorylation and that is hyperphosphorylated in several neurodegenerative diseases. Because phosphorylation regulates both normal and pathological tau functioning, it is of interest to identify the signaling pathways and enzymes capable of modulating tau phosphorylation in vivo. In SH-SY5Y human neuroblastoma cells and rat primary cortical cultures tau is phosphorylated at Ser262/356, within its microtubule-binding domain, by a staurosporine-sensitive protein kinase in response to the vicinal thiol-directed agent phenylarsine oxide. A 100-kDa protein kinase activity is present in SH-SY5Y cells that associates with microtubules, phosphorylates tau at Ser262/356, is activated by phenylarsine oxide, and is inhibited by the protein kinase inhibitor staurosporine. Isolation of individual protein bands from a polyacrylamide gel reveal two closely spaced proteins containing Ser262/356-directed protein kinase activity. These protein bands correspond to the 100-kDa microtubule/MAP-affinity regulating kinase (MARK), which phosphorylates tau within its microtubule-binding domain. Immunoblot analysis of the protein kinase bands confirm this finding, providing the first demonstration that activation of endogenous MARK results in increased tau phosphorylation within its microtubule-binding domain in situ (Jenkins, 2000).
Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate certain sites in tau protein (KXGS motifs in the repeat domain). This type of phosphorylation is enhanced in abnormal tau from Alzheimer brain tissue and causes the detachment of tau from microtubules. MARK-related kinases (PAR-1 and KIN1) occur in various organisms and are involved in establishing and maintaining cell polarity. MARK2 affects the differentiation and outgrowth of cell processes from neuroblastoma and other cell models. MARK2 phosphorylates tau protein at the KXGS motifs; this results in the detachment of tau from microtubules and their destabilization. The formation of neurites in N2a cells is blocked if MARK2 is inactivated, either by transfecting a dominant negative mutant, or by MARK2 inhibitors such as hymenialdisine. Alternatively, neurites are blocked if the target KXGS motifs on tau are rendered nonphosphorylatable by point mutations. The results suggest that MARK2 contributes to the plasticity of microtubules needed for neuronal polarity and the growth of neurites (Biernat, 2002).
MARK, a kinase family related to PAR-1 involved in establishing cell polarity, phosphorylates microtubule-associated proteins (tau/MAP2/MAP4) at KXGS motifs, causes detachment from microtubules, and their disassembly. The sites are prominent in tau from Alzheimer's disease brains. The activation of MARK was studied and the upstream kinase, MARKK, a member of the Ste20 kinase family, was identified. It phosphorylates MARK within the activation loop (T208 in MARK2). A fraction of MARK in brain tissue is doubly phosphorylated (at T208/S212), reminiscent of the activation of MAP kinase; however, the phosphorylation of the second site in MARK (S212) is inhibitory. In cells the activity of MARKK enhances microtubule dynamics through the activation of MARK and leads to phosphorylation and detachment of tau or equivalent MAPs from microtubules. Overexpression of MARK eventually leads to microtubule breakdown and cell death, but in neuronal cells the primary effect is to allow the development of neurites during differentiation (Timm, 2003).
The MARK protein kinases were originally identified by their ability to phosphorylate a serine motif in the microtubule binding domain of tau that is critical for microtubule binding. A novel human paralog, MARK4, shares 75% overall homology with MARK1-3 and is predominantly expressed in brain. Homology is most pronounced in the catalytic domain (90%); MARK4 readily phosphorylates tau and the related microtubule-associated proteins MAP2 and MAP4. In contrast to the three paralogs that all exhibit uniform cytoplasmic localization, MARK4 colocalizes with the centrosome and with microtubules in cultured cells. Overexpression of MARK4 causes thinning out of the microtubule network, concomitant with a reorganization of microtubules into bundles. In line with these findings, a tandem-affinity purified MARK4 protein complex contains alpha-, beta-, and gamma-tubulin. In differentiated neuroblastoma cells, MARK4 is localized prominently at the tips of neurite-like processes. It is suggested that, while the four MARK/PAR-1 kinases might play multiple cellular roles in concert with different targets, MARK4 is likely to be directly involved in microtubule organization in neuronal cells and may contribute to the pathological phosphorylation of tau in Alzheimers disease (Trinczek, 2003).
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