Crystal Structure of the MuSK Tyrosine Kinase

ACK1 is a multidomain non-receptor tyrosine kinase that is an effector of the Cdc42 GTPase. Members of the ACK family have a unique domain ordering and are the only tyrosine kinases known to interact with Cdc42. In contrast with many protein kinases, ACK1 has only a modest increase in activity upon phosphorylation. We have solved the crystal structures of the human ACK1 kinase domain in both the unphosphorylated and phosphorylated states. Comparison of these structures reveals that ACK1 adopts an activated conformation independent of phosphorylation. Furthermore, the unphosphorylated activation loop is structured, and its conformation resembles that seen in activated tyrosine kinases. In addition to the apo structure, complexes are also presented with a non-hydrolyzable nucleotide analog (adenosine 5 -(␤,␥-methylenetriphosphate)) and with the natural product debromohymenialdisine, a general inhibitor of many protein kinases. Analysis of these structures reveals a typical kinase fold, a pre-organization into the activated conformation, and an unusual substrate-binding cleft.ACK1 (activated Cdc42-associated kinase-1) is a member of a small family of non-receptor tyrosine kinases that bind Cdc42 in its GTP-bound form. Members of the ACK family have been identified in many species, including human (TNK1) (1), cow (ACK2) (2), Drosophila (DAck, DPR2) (3, 4), and Caenorhabditis elegans (ARK1) (5). ACK1 is a multidomain protein, and all family members contain an SH3 1 domain and a C-terminal proline-rich region. ACK family members are largely expressed in brain and skeletal tissue (2), and multiple lines of evidence suggest that they are involved in the regulation of cell adhesion and growth (6), receptor degradation (7), and axonal guidance (3). ACK1 has been implicated as a mediator of epidermal growth factor signaling to Rho family GTP-binding proteins through phosphorylation and activation of guanosine exchange factors such as Dbl, suggesting a role in regulation of the cytoskeleton (8, 9). ACK1 has also been reported to bind to adaptor proteins, suggesting that it is involved in several different signaling pathways. The SH3 domain of ACK1 binds to the proline-rich region of HSH2, an adaptor protein in hematopoietic cells, leading to its phosphorylation (10), whereas the C-terminal proline-rich domain of ACK1 interacts with the adaptor proteins Nck (11) and Grb2 (12), which are involved in cytoskeletal rearrangement (13) and Ras activation (14), respectively. In addition, both DAck and ACK2 interact with the sorting nexin SH3PX1 through their C-terminal domains, leading to its phosphorylation (3, 7), and this ACK2 interaction with SH3PX1 together with clathrin promotes degradation of EGFR (7).The domain architecture of the ACK family is unique compared with other non-receptor tyrosine kinases, and ACK family members are the only tyrosine kinases known to interact with Cdc42 (15). Members of the ACK family are specific for Cdc42 and do not bind the closely related Rac and Rho GTPases (2, 15). The 114-kDa human ACK1 p...

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“…ing (39,40). In fibroblast growth factor receptor-1, the unphosphorylated activation loop occupies the substratebinding site (41).…”

Section: Resultsmentioning

confidence: 99%

Crystal Structures of the Phosphorylated and Unphosphorylated Kinase Domains of the Cdc42-associated Tyrosine Kinase ACK1

Lougheed

Chen

Mak

et al. 2004

Journal of Biological Chemistry

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“…6. Surprisingly, this part of the STN8 sequence has a high homology to a consensus of a subfamily of protein-tyrosine kinases (TyrKc) with the known structure (47). The structural basis for selectivity of tyrosine kinases is in the peptide loops surrounding the active site and obstructing access of the shorter serine and threonine residues of the substrate proteins (48).…”

Section: Discussionmentioning

confidence: 99%

STN8 Protein Kinase in Arabidopsis thaliana Is Specific in Phosphorylation of Photosystem II Core Proteins

Vainonen¹,

Hansson

Vener

2005

Journal of Biological Chemistry

190

253

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light-exposed leaves of two Arabidopsis lines with T-DNA insertions in the stn8 gene was found significantly reduced in the assays with anti-phosphothreonine antibodies. Protein phosphorylation in each of the mutants was quantified comparatively to the wild type by mass spectrometric analyses of phosphopeptides released from the photosynthetic membranes and differentially labeled with stable isotopes. The lack of STN8 caused 50 -60% reduction in D1 and D2 phosphorylation, but did not change the phosphorylation level of two peptides that could correspond to light-harvesting proteins encoded by seven different genes in Arabidopsis. Phosphorylation of the PsbH protein at Thr-4 was completely abolished in the plants lacking STN8. Phosphorylation of Thr-4 in the wild type required both light and prior phosphorylation at Thr-2, indicating that STN8 is a light-activated kinase that phosphorylates Thr-4 only after another kinase phosphorylates Thr-2. Analysis of the STN8 catalytic domain suggests that selectivity of STN8 in phosphorylation of the very N-terminal residues in D1, D2, and CP43, and Thr-4 in PsbH pre-phosphorylated at Thr-2 may be explained by the long loops obstructing entrance into the kinase active site and seven additional basic residues in the vicinity of the catalytic site, as compared with the homologous STN7 kinase responsible for phosphorylation of light-harvesting proteins.Reversible protein phosphorylation is a key molecular mechanism regulating all aspects of physiology and development in eukaryotic cells. The genome sequencing of Arabidopsis thaliana has uncovered more than 1160 genes encoding for the protein kinases and phosphatases in this model plant (1), highlighting a new challenge in revealing of the substrates and functions for these catalysts of reversible protein phosphorylation. The task of identification of in vivo substrate specificities for individual protein kinases and phosphatases in Arabidopsis thaliana has recently became feasible because of two groundbreaking developments. First, the plant lines with knockouts of individual genes became public and commercially available (2). Second, new mass spectrometrybased analytical techniques permitted identification and mapping of in vivo phosphorylation sites in numerous proteins (as reviewed in Ref.3). For the first time these technical advancements provide the possibility of unraveling the complex protein phosphorylation network in plant photosynthetic membranes, which regulates the adaptation of the photosynthetic apparatus and efficient energy utilization in response to light quality and intensity, ambient temperature, circadian rhythm, nutrient deficiency, and other stresses (4 -6).The major proteins undergoing reversible phosphorylation in the photosynthetic thylakoid membranes belong to photosystem II (PSII) 3 and its light-harvesting antennae proteins LHCII (6 -11). Phosphorylation status of these proteins is differentially regulated by light (12), redox state of the membrane electron carriers (13-15), the ferredoxin-thioredoxi...

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“…1A). Investigations of the PDGF, Eph, and MuSK receptors point to an additional, autoinhibitory role for the JMD (41,42,(52)(53)(54). Taking into account this possibility, we made three point mutants.…”

Section: Mutation Of Tyrmentioning

confidence: 99%

“…Y559F Displays Decreased Tyrosine Phosphorylation Independent of Src Kinase Activity-In addition to serving as a binding site for interacting proteins, the JMD of RTKs may also function in kinase regulation (41)(42)(43)(44)52). To investigate this hypothesis, we examined several indicators of receptor kinase activity.…”

Section: Mutation Of Tyrmentioning

confidence: 99%

A Juxtamembrane Tyrosine in the Colony Stimulating Factor-1 Receptor Regulates Ligand-induced Src Association, Receptor Kinase Function, and Down-regulation

Rohde

Schrum

Lee

2004

Journal of Biological Chemistry

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Crystal Structure of the MuSK Tyrosine Kinase

Cited by 127 publications

References 45 publications

Crystal Structures of the Phosphorylated and Unphosphorylated Kinase Domains of the Cdc42-associated Tyrosine Kinase ACK1

Crystal Structures of the Phosphorylated and Unphosphorylated Kinase Domains of the Cdc42-associated Tyrosine Kinase ACK1

STN8 Protein Kinase in Arabidopsis thaliana Is Specific in Phosphorylation of Photosystem II Core Proteins

A Juxtamembrane Tyrosine in the Colony Stimulating Factor-1 Receptor Regulates Ligand-induced Src Association, Receptor Kinase Function, and Down-regulation

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