Dynamin Is a Minibrain Kinase/Dual Specificity Yak1-related Kinase 1A Substrate

Abstract

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.Minibrain kinase (Mnbk) 1 was originally identified in Drosophila as a mutation affecting neurogenesis (1). Mnbk mutant flies, which have low levels of kinase expression, possess fewer neuroblasts and a reduced brain volume as compared with the wild type (WT), especially in the optic lobes and central brain (1). The reduction in brain size leads to several distinct learning and behavioral defects. Because the Mnbk mutation does not appear to affect the development of Drosophila until late in the third instar, it was postulated that the Mnbk gene is required for the proliferation of neuroblasts during postembryonic neurogenesis (1).Dual specificity Yak 1-related kinase (Dyrk) 1A was subsequently cloned (2-5) and identified as the mammalian homologue of the Drosophila Mnbk gene. The Mnbk/Dyrk1A gene is a member of a growing family of Dyrk-related genes (6) whose members include Yak1 (7), several Dyrks (8), ANPK (9), HIPK2 (10), Mirk (11), Myak (12), and Pom1p (13). Mammalian Mnbk/Dyrk1A gene contains either 763 or 754 amino acid residues as a result of alternative splicing (2). The kinase domain of Mnbk/Dyrk1A, consisting of ϳ320 residues, is...

Phosphorylation of Ser640 in Muscle Glycogen Synthase by DYRK Family Protein Kinases

“…Mice heterozygous for the mutation (DYRK1A ϩ/Ϫ ) had a decreased brain size in a region-specific manner (32). DYRK1A phosphorylates various substrates in vitro, including the signal transducer and activator of transcription 3 (STAT3) (33), the ⑀ subunit of eukaryotic initiation factor 2B, the microtubuleassociated protein tau (34), the transcription factor of the forkhead family FKHR (35), and dynamin (36) indicating that DYRK1A might participate in several biochemical pathways. (Fig.…”

Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) Modulates Serine/Arginine-rich Protein 55 (SRp55)-promoted Tau Exon 10 Inclusion

“…Plasmids, Proteins, and Antibodies-Recombinant rat Dyrk1A and mammalian expression vector pcDNA3 containing either rat Dyrk1A or dominant negative Dyrk1A K188R were kindly provided by Dr. Y.-W. Hwang of the New York State Institute for Basic Research and prepared as described previously (17). pCEP4/ SRp55-HA was a gift from Dr. Tarn of the Institute of Biomedical Sciences, Academia Sinica, Taiwan.…”

“…One microgram of total RNA was used for first strand cDNA synthesis with oligo(dT) [15][16][17][18] by using an Omniscript reverse transcription kit (Qiagen). PCR was performed by using PrimeSTAR TM HS DNA polymerase (Takara Bio Inc., Otsu, Shiga, Japan) with primers (forward, 5Ј-GGT-GTCCACTCCCAGTTCAA-3Ј; and reverse, 5Ј-CCCTGGTT-TATGATGGATGTTG CCTAATGAG-3Ј) to measure alternative splicing of Tau exon 10 under conditions: 98°C for 3 min, 98°C for 10 s, and 68°C for 40 s for 30 cycles, followed by 68°C for 10 min for extension.…”

On dendrites in Down syndrome and DS murine models: a spiny way to learn

“…Dyrk1A also regulates the development of dendritic trees of neurones and modulates the activity of the c-AMP response elementbinding protein (CREB), which participates in signal transduction pathways involved in synaptic plasticity and neuronal differentiation (Hammerle et al, 2003). Finally, DYRK kinases may modulate dendrite development by regulating vesicle trafficking which is dependent on Dynamin (DYN1), a GTPase putative substrate of DYRK1A, that plays a fundamental role in synaptic vesicle recycling, clathrin-mediated endocytosis, intracellular membrane trafficking (see Torre et al, 1994 for a review) and neurite outgrowth (Chen-Hwang et al, 2002;Kim et al, 1997). DYRK1A is transported through the growing dendritic tree where it co-localizes with DYN1 (Hammerle et al, 2003).…”