The role of overexpressed DYRK1A protein in the early onset of neurofibrillary degeneration in Down syndrome.

The gene encoding the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A (DYRK1A) is located in the Down syndrome (DS) critical region of chromosome 21. The third copy of DYRK1A is believed to contribute to abnormal brain development in patients with DS. In vitro studies showing that DYRK1A phosphorylates tau protein suggest that this kinase is also involved in tau protein phosphorylation in the human brain and contributes to neurofibrillary degeneration, and that this contribution might be enhanced in patients with DS. To explore this hypothesis, the brain tissue from 57 subjects including 16 control subjects, 21 patients with DS, and 20 patients with sporadic Alzheimer's disease (AD) was examined with two antibodies to the amino-terminus of DYRK1A (7F3 and G-19), as well as two polyclonal antibodies to its carboxy-terminus (X1079 and 324446). Western blots demonstrated higher levels of full-length DYRK1A in the brains of patients with DS when compared to control brains. Immunocytochemistry revealed that DYRK1A accumulates in neurofibrillary tangles (NFTs) in subjects with sporadic AD and in subjects with DS/AD. Overexpression of DYRK1A in patients with DS was associated with an increase in DYRK1A-positive NFTs in a gene dosage-dependent manner. Results support the hypothesis that overexpressed DYRK1A contributes to neurofibrillary degeneration in DS more significantly than in subjects with two copies of the DYRK1A gene and sporadic AD. Immunoreactivity with antibodies against DYRK1A not only in NFTs but also in granules in granulovacuolar degeneration and in corpora amylacea suggests that DYRK1A is involved in all three forms of degeneration and that overexpression of this kinase may contribute to the early onset of these pathologies in DS.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A does not require tyrosine phosphorylation for activity in vitro.

The dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) gene is localized in human chromosome 21, and its overexpression has been associated with the learning and memory deficits of Down syndrome. DYRK1A contains a Y319XY321 motif shared by all members of the DYRK protein kinase family. Residue Y321 in the motif is phosphorylated in DYRK1A prepared from Escherichia coli and from eukaryotic cells. It has been proposed that the YXY motif is an equivalent of the TXY motif, the activation loop, of mitogen-activated protein kinase and that phosphorylation at the motif is required for DYRK activity. In this study, the role of tyrosine phosphorylation in the activity of DYRK1A was investigated in detail. Wild-type DYRK1A with a reduced level of phosphotyrosine (pY) was prepared by treating E. coli-produced DYRK1A with two different protein tyrosine phosphatases. The resulting pY-depleted DYRK1A could not regain pY during autophosphorylation but was as active as the untreated control. These findings were further supported by the observation that DYRK1A retained significant enzymatic activity when both tyrosine residues in the YXY motif were replaced with either histidine or glutamine. Together, we conclude that tyrosine phosphorylation and tyrosine residues in the YXY motif are not directly involved in DYRK1A enzymatic activity in vitro.

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.

Kinetic properties of a MNB/DYRK1A mutant suitable for the elucidation of biochemical pathways.

Minibrain kinase/dual-specificity tyrosine phosphorylation regulated kinase 1A (MNB/DYRK1A) is a proline/arginine-directed serine/threonine kinase implicated in the learning deficits of Down syndrome. Epigallocatechin-3-gallate (EGCG), the major tea polyphenolic compound, is a potent MNB/DYRK1A inhibitor. In this study, we investigated the mechanism of EGCG inhibition of MNB/DYRK1A using a combination of genetic and biochemical approaches. In the testing system using MNB/DYRK1A-promoted Gli 1-dependent transcription as the readout, NIH3T3 cells expressing EGCG resistant MNB/DYRK1A mutant R21 were found to acquire EGCG resistance for a wide range of drug concentrations. Mutant R21 harbors a single K465R substitution, which produces a 3-fold gain in the EGCG resistance in vitro. However, the gain in the EGCG resistance alone cannot fully interpret the effectiveness of mutant R21 in suppressing EGCG in cultured cells. Kinetic analysis suggests that EGCG functions as a noncompetitive inhibitor against ATP. Interestingly, the K465R mutation changes the mode of EGCG inhibition on MNB/DYRK1A so that it becomes a competitive inhibitor against ATP. This competitive mode of EGCG inhibition coupled with high intracellular ATP concentrations and an elevated EGCG resistance are likely to be the basis for the resistant property of mutant R21 in cultured cells. The K465R mutation apparently transforms the intramolecular interactions required for MNB/DYRK1A catalysis. This mutant would also be valuable for the elucidation of the mechanisms of MNB/DYRK1A-catalyzed reaction.

Phosphorylation of amphiphysin I by minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase, a kinase implicated in Down syndrome.

Minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase (Mnb/Dyrk1A) is a proline-directed serine/threonine kinase encoded in the Down syndrome critical region of human chromosome 21. This kinase has been shown to phosphorylate dynamin 1 and synaptojanin 1. Here we report that amphiphysin I (Amph I) is also a Mnb/Dyrk1A substrate. This kinase phosphorylated native Amph I in rodent brains and recombinant human Amph I expressed in Escherichia coli. Serine 293 (Ser-293) was identified as the major site, whereas serine 295 and threonine 310 were found as minor kinase sites. In cultured cells, recombinant Amph I was phosphorylated at Ser-293 by endogenous kinase(s). Because mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) has been suggested to phosphorylate Amph I at Ser-293, our efforts addressed whether Ser-293 is phosphorylated in vivo by MAPK/ERK or by Mnb/Dyrk1A. Overnight serum-withdrawal inactivated MAPK/ERK; nonetheless, Ser-293 was phosphorylated in Chinese hamster ovary and SY5Y cells. Epigallocatechin-3-gallate, a potent Mnb/Dyrk1A inhibitor in vitro, apparently reduced the phosphorylation at Ser-293, whereas PD98059, a potent MAPK/ERK inhibitor, did not. High frequency stimulation of mouse hippocampal slices reduced the phosphorylation at Ser-293, albeit in the midst of MAPK/ERK activation. The endophilin binding in vitro was inhibited by phosphorylating Amph I with Mnb/Dyrk1A. However, phosphorylation at Ser-293 did not appear to alter cellular distribution patterns of the protein. Our results suggest that Mnb/Dyrk1A, not MAPK/ERK, is responsible for in vivo phosphorylation of Amph I at Ser-293 and that phosphorylation changes the recruitment of endophilin at the endocytic sites.

Mnb/Dyrk1A phosphorylation regulates the interaction of dynamin 1 with SH3 domain-containing proteins.

Mnb/Dyrk1A is a proline-directed serine/threonine kinase implicated in Down's syndrome. Mnb/Dyrk1A was shown to phosphorylate dynamin 1 and alter its interactions with several SH3 domain-containing endocytic accessory proteins. To determine the mechanism of regulation, we mapped the Mnb/Dyrk1A phosphorylation sites in dynamin 1. Using a combination of deletion mutants and synthetic peptides, three potential Mnb/Dyrk1A phosphorylation sites (S778, S795, and S857) were first identified. Phosphorylation at S795 and S857 was confirmed in full-length dynamin 1, and S857 was subsequently determined to be the major Mnb/Dyrk1A phosphorylation site in vitro. Phosphorylation at S857 was demonstrated to be the basis for altering the binding of dynamin 1 to amphiphysin 1 and Grb 2 by site-directed mutants mimicking phosphorylation. Furthermore, S857 of dynamin 1 is phosphorylated by the endogenous kinase in brain extracts and in PC12 cells. In PC12 cells, the state of S857 phosphorylation is dependent on membrane potentials. These results suggest that S857 phosphorylation is a physiological event, which regulates the binding of dynamin 1 to SH3 domain-containing proteins. Since S857 is unique to dynamin 1xa isoforms, Mnb/Dyrk1A regulation of dynamin 1 is expected to be specific to these spliced variants.

Cell type- and brain structure-specific patterns of distribution of minibrain kinase in human brain.

The minibrain kinase (Mnb/Dyrk1A) gene is localized in the Down syndrome (DS) critical region of chromosome 21. This gene encodes a proline-directed serine/threonine protein kinase (minibrain kinase-Mnb/Dyrk1A), which is required for the proliferation of distinct neuronal cell types during postembryonic neurogenesis. To study the distribution of Mnb/Dyrk1A during human brain development and aging, we raised Mnb/Dyrk1A-specific antibody (mAb 7F3) and examined 22 brains of normal subjects from 8 months to 90 years of age. We found that neurons were the only cells showing the presence of 7F3-positive product in both cell nucleus and cytoplasm. Nuclear localization supports the concept that Mnb/Dyrk1A may be involved in control of gene expression. Synaptic localization of Mnb/Dyrk1A also supports our previous studies suggesting that Mnb/Dyrk1A is a regulator of assembly of endocytic apparatus and appears to be involved in synaptic vesicle recycling and synaptic signal transmission. Accumulation of numerous 7F3-positive corpora amylacea in the memory and motor system subdivisions in subjects older than 33 years of age indicates that Mnb/Dyrk1A is colocalized with markers of astrocyte and neuron degeneration. Differences in the topography and the amount of Mnb/Dyrk1A in neurons, astrocytes, and ependymal and endothelial cells appear to reflect cell type- and brain structure-specific patterns in trafficking and utilization of Mnb/Dyrk1A.

Dynamin is a minibrain kinase/dual specificity Yak1-related kinase 1A substrate.

The minibrain kinase (Mnbk)/dual specificity Yak 1-related kinase 1A (Dyrk1A) gene is implicated in the mental retardation associated with Down's syndrome. It encodes a proline-directed serine/threonine kinase whose function has yet to be defined. We have used a solid-phase Mnbk/Dyrk1A kinase assay to aid in the search for the cellular Mnbk/Dyrk1A substrates. The assay revealed that rat brain contains two cytosolic proteins, one with a molecular mass of 100 kDa and one with a molecular mass of 140 kDa, that were prominently phosphorylated by Mnbk/Dyrk1A. The 100-kDa protein was purified and identified as dynamin 1. The conclusion was further supported by evidence that a recombinant glutathione S-transferase fusion protein containing dynamin isoform 1aa was phosphorylated by Mnbk/Dyrk1A. In addition to isoform 1aa, Mnbk/Dyrk1A also phosphorylated isoforms 1ab and 2aa but not human MxA protein when analyzed by the solid-phase kinase assay. Upon Mnbk/Dyrk1A phosphorylation, the interaction of dynamin 1 with the Src homology 3 domain of amphiphysin 1 was reduced. However, when Mnbk/Dyrk1A phosphorylation was allowed to proceed more extensively, the phosphorylation enhanced rather than reduced the binding of dynamin 1 to amphiphysin 1. The result suggests that Mnbk/Dyrk1A can play a dual role in regulating the interaction of dynamin 1 with amphiphysin 1. Mnbk/Dyrk1A phosphorylation also reduced the interaction of dynamin with endophilin 1, whereas the same phosphorylation enhanced the binding of dynamin 1 to Grb2. Nevertheless, the dual function of Mnbk/Dyrk1A phosphorylation was not observed for the interaction of dynamin 1 with endophilin 1 or Grb2. The interactions of dynamin with amphiphysin and endophilin are essential for the formation of endocytic complexes; our results suggest that Mnbk/Dyrk1A may function as a regulator controlling the assembly of endocytic apparatus.