Roles of the DYRK kinase Pom2 in cytokinesis, mitochondrial morphology, and sporulation in fission yeast

Pom2 is predicted to be a dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) related to Pom1 in Schizosaccharomyces pombe. DYRKs share a kinase domain capable of catalyzing autophosphorylation on tyrosine and exogenous phosphorylation on serine/threonine residues. Here we show that Pom2 is functionally different from the well-characterized Pom1, although they share 55% identity in the kinase domain and the Pom2 kinase domain functionally complements that of Pom1. Pom2 localizes to mitochondria throughout the cell cycle and to the contractile ring during late stages of cytokinesis. Overexpression but not deletion of pom2 results in severe defects in cytokinesis, indicating that Pom2 might share an overlapping function with other proteins in regulating cytokinesis. Gain and loss of function analyses reveal that Pom2 is required for maintaining mitochondrial morphology independently of microtubules. Intriguingly, most meiotic pom2Δ cells form aberrant asci with meiotic and/or forespore membrane formation defects. Taken together, Pom2 is a novel DYRK kinase involved in regulating cytokinesis, mitochondrial morphology, meiosis, and sporulation in fission yeast.     

Specificity determinants of substrate recognition by the protein kinase DYRK1A

DYRK1A is a dual-specificity protein kinase that is thought to be involved in brain development. We identified a single phosphorylated amino acid residue in the DYRK substrate histone H3 (threonine 45) by mass spectrometry, phosphoamino acid analysis, and protein sequencing. Exchange of threonine 45 for alanine abolished phosphorylation of histone H3 by DYRK1A and by the related kinases DYRK1B, DYRK2, and DYRK3 but not by CLK3. In order to define the consensus sequence for the substrate specificity of DYRK1A, a library of 300 peptides was designed in variation of the H3 phosphorylation site. Evaluation of the phosphate incorporation into these peptides identified DYRK1A as a proline-directed kinase with a phosphorylation consensus sequence (RPX(S/T)P) similar to that of ERK2 (PX(S/T)P). A peptide designed after the optimal substrate sequence (DYRKtide) was efficiently phosphorylated by DYRK1A (K(m) = 35 microM) but not by ERK2. Both ERK2 and DYRK1A phosphorylated myelin basic protein, whereas only ERK2, but not DYRK1A, phosphorylated the mitogen-activated protein kinase substrate ELK-1. This marked difference in substrate specificity between DYRK1A and ERK2 can be explained by the requirement for an arginine at the P -3 site of DYRK substrates and its presumed interaction with aspartate 247 conserved in all DYRKs.     

MNB/DYRK1A phosphorylation regulates the interactions of synaptojanin 1 with endocytic accessory proteins

MNB/DYRK1A is a proline-directed serine/threonine kinase implicated in Down syndrome (DS). In an earlier screening, two proteins from adult rat brain, one 100kDa and the other 140 kDa, were found to be prominently phosphorylated by the kinase. The 100-kDa protein was previously characterized as an isoform of dynamin 1. In this study, we identified the 140-kDa protein as synaptojanin 1 (SJ1). MNB/DYRK1A phosphorylates SJ1 at multiple sites and produces complex behaviors in binding to amphiphysin 1 and intersectin 1 (ITSN1). However, the phosphorylation has little effect on the phosphatidylinositol phosphatase activity of SJ1. These results suggest that MNB/DYRK1A is involved in regulating the recruitment activity but not the phosphatase activity of SJ1. Our findings may be especially important in the etiology of DS because MNB/DYRK1A, SJ1, and ITSN1 are all located at or near the region of human chromosome 21, which is postulated to be involved in the disease.     

The DYRK-family kinase Pom1 phosphorylates the F-BAR protein Cdc15 to prevent division at cell poles

Division site positioning is critical for both symmetric and asymmetric cell divisions. In many organisms, positive and negative signals cooperate to position the contractile actin ring for cytokinesis. In rod-shaped fission yeast Schizosaccharomyces pombe cells, division at midcell is achieved through positive Mid1/anillin-dependent signaling emanating from the central nucleus and negative signals from the dual-specificity tyrosine phosphorylation-regulated kinase family kinase Pom1 at the cell poles. In this study, we show that Pom1 directly phosphorylates the F-BAR protein Cdc15, a central component of the cytokinetic ring. Pom1-dependent phosphorylation blocks Cdc15 binding to paxillin Pxl1 and C2 domain protein Fic1 and enhances Cdc15 dynamics. This promotes ring sliding from cell poles, which prevents septum assembly at the ends of cells with a displaced nucleus or lacking Mid1. Pom1 also slows down ring constriction. These results indicate that a strong negative signal from the Pom1 kinase at cell poles converts Cdc15 to its closed state, destabilizes the actomyosin ring, and thus promotes medial septation.     

Pom1 DYRK regulates localization of the Rga4 GAP to ensure bipolar activation of Cdc42 in fission yeast

BACKGROUND: In the fission yeast Schizosaccharomyces pombe, cell growth takes place exclusively at both ends of the cylindrical cell. During this highly polarized growth, microtubules are responsible for the placement of the cell-end marker proteins, the Tea1-Tea4/Wsh3 complex, which recruits the Pom1 DYRK-family protein kinase. Pom1 is required for proper positioning of growth sites, and the Deltapom1 mutation brings about monopolar cell growth. RESULTS: Pom1 kinase physically interacts with Rga4, which has a GAP (GTPase-activating protein) domain for Rho-family GTPase. Genetic and biochemical evidence indicates that Rga4 functions as GAP for the Cdc42 GTPase, an evolutionarily conserved regulator of F-actin. CRIB (Cdc42/Rac interactive binding)-GFP microscopy has revealed that GTP-bound, active Cdc42 is concentrated to growing cell ends accompanied by developed F-actin structures, where the Rga4 GAP is excluded. The monopolar Deltapom1 mutant fails to eliminate Rga4 from the nongrowing cell end, resulting in monopolar distribution of GTP-Cdc42 to the growing cell end. However, mutational inactivation of Rga4 allows Cdc42 to be active at both ends of Deltapom1 cells, suggesting that mislocalization of Rga4 in the Deltapom1 mutant contributes to its monopolar phenotype. CONCLUSIONS: Pom1 kinase recruited to cell ends by the Tea1-Tea4/Wsh3 complex is essential for proper localization of a GAP for Cdc42, Rga4, which ensures bipolar localization of GTP-bound, active Cdc42. Because of the established role of Cdc42 in F-actin formation, these observations provide a new insight into how the microtubule system achieves localized formation of F-actin to generate cell polarity.     

The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1 and Cyclin D1 and induces cell cycle exit and neuronal differentiation

A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G₁ phase. Sustained overexpression of DYRK1A induced G₀ cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27^Kip1 on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27^Kip1 Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome.     

Protein kinase C phosphorylation of purified Na,K-ATPase: C-terminal phosphorylation sites at the alpha- and gamma-subunits close to the inner face of the plasma membrane

The alpha-subunit of the Na,K-ATPase is phosphorylated at specific sites by protein kinases A and C. Phosphorylation by protein kinase C (PKC) is restricted to the N terminus and takes place to a low stoichiometry, except in rat. Here we show that the alpha-subunit of shark Na,K-ATPase can be phosphorylated by PKC at C-terminal sites to stoichiometric levels in the presence of detergents. Two novel phosphorylation sites are possible candidates for this PKC phosphorylation: Thr-938 in the M8/M9 loop located very close to the PKA site, and Ser-774, in the proximal part of the M5/M6 hairpin. Both sites are highly conserved in all known alpha-subunits, indicating a physiological role. A similar pattern of detergent-mediated phosphorylation by PKC was found in pig kidney Na,K-ATPase alpha-subunit. Interestingly, the kidney-specific gamma-subunit was phosphorylated by PKC in the presence of detergent. The close proximity of the novel PKC sites to the membrane suggests that targeting proteins to tether PKC into the membrane phase is important in controlling the in vivo phosphorylation of this novel class of membrane-adjacent PKC sites. It is suggested that in purified preparations where functional targeting may be impaired detergents are needed to expose the sites.     

Fission yeast Pom1p kinase activity is cell cycle regulated and essential for cellular symmetry during growth and division

Schizosaccharomyces pombe cells grow from both ends during most of interphase and divide symmetrically into two daughter cells. The pom1 gene, encoding a member of the Dyrk family of protein kinases, has been identified through a mutant showing abnormal cellular morphogenesis. Here we show that Pom1p kinase activity is cell cycle regulated in correlation with the state of cellular symmetry: the activity is high during symmetrical growth and division, but lower when cells grow at just one end. Point mutations in the catalytic domain lead to asymmetry during both cell growth and division, whilst cells overexpressing Pom1p form additional growing ends. Manipulations of kinase activity indicate a negative role for Pom1p in microtubule growth at cell ends. Pom1p is present in a large protein complex and requires its non-catalytic domain to localize to the cell periphery and its kinase activity to localize to cell ends. These data establish that Pom1p kinase activity plays an important role in generating cellular symmetry and suggest that there may be related roles of homologous protein kinases ubiquitously present in all eukaryotes.     

Emerging role of DYRK family protein kinases as regulators of protein stability in cell cycle control

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell proliferation and differentiation. Members of the DYRK family phosphorylate many substrates, including critical regulators of the cell cycle. A recent report revealed that human DYRK2 acts as a negative regulator of G₁/S transition by phosphorylating c-Jun and c-Myc, thereby inducing ubiquitination-mediated degradation. Other DYRKs also function as cell cycle regulators by modulating the turnover of their target proteins. DYRK1B can induce reversible cell arrest in a quiescent G₀ state by targeting cyclin D1 for proteasomal degradation and stabilizing p27^Kip1. The DYRK2 ortholog of C. elegans, MBK-2, triggers the proteasomal destruction of oocyte proteins after meiosis to allow the mitotic divisions in embryo development. This review summarizes the accumulating results that provide evidence for a general role of DYRKs in the regulation of protein stability.     

Hierarchical disabled-1 tyrosine phosphorylation in Src family kinase activation and neurite formation

There are two developmentally regulated alternatively spliced forms of Disabled-1 (Dab1) in the chick retina: an early form (Dab1-E) expressed in retinal precursor cells and a late form (Dab1-L) expressed in neuronal cells. The main difference between these two isoforms is the absence of two Src family kinase (SFK) recognition sites in Dab1-E. Both forms retain two Abl/Crk/Nck recognition sites implicated in the recruitment of SH2 domain-containing signaling proteins. One of the Dab1-L-specific SFK recognition sites, at tyrosine(Y)-198, has been shown to be phosphorylated in Reelin-stimulated neurons. Here, we use Reelin-expressing primary retinal cultures to investigate the role of the four Dab1 tyrosine phosphorylation sites on overall tyrosine phosphorylation, Dab1 phosphorylation, SFK activation and neurite formation. We show that Y198 is essential but not sufficient for maximal Dab1 phosphorylation, SFK activation and neurite formation, with Y232 and Y220 playing particularly important roles in SFK activation and neuritogenesis, and Y185 having modifying effects secondary to Y232 and Y220. Our data support a role for all four Dab1 tyrosine phosphorylation sites in mediating the spectrum of activities associated with Reelin-Dab1 signaling in neurons.     

Emerging role of DYRK family protein kinases as regulators of protein stability in cell cycle control

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell proliferation and differentiation. Members of the DYRK family phosphorylate many substrates, including critical regulators of the cell cycle. A recent report revealed that human DYRK2 acts as a negative regulator of G₁/S transition by phosphorylating c-Jun and c-Myc, thereby inducing ubiquitination-mediated degradation. Other DYRKs also function as cell cycle regulators by modulating the turnover of their target proteins. DYRK1B can induce reversible cell arrest in a quiescent G₀ state by targeting cyclin D1 for proteasomal degradation and stabilizing p27^Kip1. The DYRK2 ortholog of C. elegans, MBK-2, triggers the proteasomal destruction of oocyte proteins after meiosis to allow the mitotic divisions in embryo development. This review summarizes the accumulating results that provide evidence for a general role of DYRKs in the regulation of protein stability.     

G protein-coupled receptor-mediated phosphorylation of the activation loop of protein kinase D: dependence on plasma membrane translocation and protein kinase Cepsilon

Protein kinase D (PKD) is a serine/threonine protein kinase activated by G protein-coupled receptor (GPCR) agonists through an incompletely characterized mechanism that includes its reversible plasma membrane translocation and activation loop phosphorylation via a protein kinase C (PKC)-dependent pathway. To gain a better understanding of the mechanism regulating the activation of PKD in response to GPCR stimulation, we investigated the role of its rapid plasma membrane translocation on its activation loop phosphorylation and identified the endogenous PKC isozyme that mediates that event in vivo. We had found that the activation loop of a PKD mutant, with reduced affinity for diacylglycerol and phorbol esters, was only phosphorylated upon its plasma membrane association. We also found that the activation loop phosphorylation and rapid plasma membrane dissociation of PKD were inhibited either by preventing the plasma membrane translocation of PKCepsilon, through abolition of its interaction with receptor for activated C kinase, or by suppressing the expression of PKCepsilon via specific small interfering RNAs. Thus, this study demonstrates that the plasma membrane translocation of PKD, in response to GPCR stimulation, is necessary for the PKCepsilon-mediated phosphorylation of the activation loop of PKD and that this event requires the translocation of both kinases to the plasma membrane. Based on these and previous results, we propose a model of GPCR-mediated PKD regulation that integrates its changes in distribution, catalytic activity, and multisite phosphorylation.     

Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation.

A central feature of signal transduction downstream of both receptor and oncogenic tyrosine kinases is the Ras-dependent activation of a protein kinase cascade consisting of Raf-1, Mek (MAP kinase kinase) and ERKs (MAP kinases). To study the role of tyrosine kinase activity in the activation of Raf-1, we have examined the properties of p74Raf-1 and oncogenic Src that are necessary for activation of p74Raf-1. We show that in mammalian cells activation of p74Raf-1 by oncogenic Src requires pp60Src to be myristoylated and the ability of p74Raf-1 to interact with p21Ras-GTP. The Ras/Raf interaction is required for p21Ras-GTP to bring p74Raf-1 to the plasma membrane for phosphorylation at tyrosine 340 or 341, probably by membrane-bound pp60Src. When oncogenic Src is expressed with Raf-1, p74Raf-1 is activated 5-fold; however, when co-expressed with oncogenic Ras and Src, Raf-1 is activated 25-fold and this is associated with a further 3-fold increase in tyrosine phosphorylation. Thus, p21Ras-GTP is the limiting component in bringing p74Raf-1 to the plasma membrane for tyrosine phosphorylation. Using mutants of Raf-1 at Tyr340/341, we show that in addition to tyrosine phosphorylation at these sites, there is an additional activation step resulting from p21Ras-GTP recruiting p74Raf-1 to the plasma membrane. Thus, the role of Ras in Raf-1 activation is to bring p74Raf-1 to the plasma membrane for at least two different activation steps.     

Sprouty2-mediated inhibition of fibroblast growth factor signaling is modulated by the protein kinase DYRK1A

Raf-MEK-extracellular signal-regulated kinase (Erk) signaling initiated by growth factor-engaged receptor tyrosine kinases (RTKs) is modulated by an intricate network of positive and negative feedback loops which determine the specificity and spatiotemporal characteristics of the intracellular signal. Well-known antagonists of RTK signaling are the Sprouty proteins. The activity of Sprouty proteins is modulated by phosphorylation. However, little is known about the kinases responsible for these posttranslational modifications. We identify DYRK1A as one of the protein kinases of Sprouty2. We show that DYRK1A interacts with and regulates the phosphorylation status of Sprouty2. Moreover, we identify Thr75 on Sprouty2 as a DYRK1A phosphorylation site in vitro and in vivo. This site is functional, since its mutation enhanced the repressive function of Sprouty2 on fibroblast growth factor (FGF)-induced Erk signaling. Further supporting the idea of a functional interaction, DYRK1A and Sprouty2 are present in protein complexes in mouse brain, where their expression overlaps in several structures. Moreover, both proteins copurify with the synaptic plasma membrane fraction of a crude synaptosomal preparation and colocalize in growth cones, pointing to a role in nerve terminals. Our results suggest, therefore, that DYRK1A positively regulates FGF-mitogen-activated protein kinase signaling by phosphorylation-dependent impairment of the inhibitory activity of Sprouty2.     

Neuronal transmission stimulates the phosphorylation of Kv1.4 channel at Ser229 through protein kinase A1

Phosphorylation of voltage-gated K+ channels (Kv) is involved in regulation of neuronal excitability, synaptic plasticity and neuronal survival. Among Kv channels expressed in the CNS, Kv1.4 is located in the soma, dendrite and axon terminus of neurones in most regions of the brain. Here, we show that Ser229 found within the highly conserved T1 domain of Kv1.4 in cultured rat cortical neurones is phosphorylated by protein kinase A (PKA), as demonstrated by in vitro protein kinase assay and Western blotting with a polyclonal antibody specific against phosphorylated Ser229. Glutamate, high concentrations of K+ or K+ channel blockers known to increase neurotransmission all stimulated the phosphorylation of Kv1.4 at Ser229 via N-methyl-D-aspartate (NMDA), but not alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptor, whereas tetradotoxin (TTX), known to block neuronal transmission, and depletion of extracellular Ca2+ inhibited phosphorylation induced by tetraethylammonium (TEA), a non-selective K+ channel blocker. Mutation of Ser229 to Ala229 enhanced the current density. Taken together, elevation of the neuronal transmission stimulates the phosphorylation of Kv1.4 at Ser229 via the Ca2+ influx through NMDA receptor. Thus, it is possible that neuronal transmission regulates neuronal excitability partially through the phosphorylation of Kv1.4S229.