Keywords
CASK, MAGUK, pseudokinase, Mg²⁺-independent kinase, neurexin, PDZ domain, DFG-motif, GFG-motif, autophosphorylation, CaM-kinase domain, ATP binding, kinase structure
Reference
DOI: 10.1016/j.cell.2008.02.036
Abstract Summary
- CASK, a MAGUK protein, contains an N-terminal CaM-kinase domain, previously thought catalytically inactive (pseudokinase) due to absence of DFG motif for Mg²⁺-binding.
- This study shows CASK is catalytically active without Mg²⁺, phosphorylating itself and neurexin-1, its synaptic partner.
- High-res crystal structures reveal a constitutively active kinase conformation, ATP binding, and Mg²⁺-independent phosphotransfer.
- CASK bridges scaffolding (MAGUK) and catalytic (kinase) roles, and highlights that other “pseudokinases” may also possess latent kinase activity.
1. CASK Structure & Domain Organization
| Domain | Function |
|---|---|
| N-terminal CaM-kinase domain | Unusual kinase domain lacking DFG, catalytically active without Mg²⁺. |
| L27 domain | Protein-protein interactions. |
| PDZ domain | Binds C-terminal PDZ-binding motifs of partners like neurexin-1. |
| SH3 domain | Scaffold, mediating intramolecular and intermolecular contacts. |
| Guanylate kinase (GK) domain | Scaffolding, mediating complex formation. |
CASK integrates kinase activity with scaffolding roles, organizing synaptic complexes.
2. CASK CaM-Kinase Domain: Unusual Catalytic Mechanism
- Crystal structures (3C0G, 3C01) of CASK’s kinase domain (residues 1-337):
- Typical kinase fold: N-terminal lobe (β-sheets), C-terminal lobe (α-helices).
- Closed, active-like conformation resembling DAPK1-ATP complex, distinct from inactive CaMKI.
- Key structural features:
- Gly-rich loop (residues 19–24).
- Lys-Glu salt bridge (Lys41-Glu62) for ATP positioning.
- Catalytic loop: Asp141 (catalytic), Cys146 (instead of conserved Asn, interacts with Asp141).
- Activation segment: ordered, enabling substrate binding.
- DFG motif mutated to GFG (Asp162→Gly), preventing Mg²⁺ binding.
3. ATP and Substrate Binding
| Aspect | Observation in CASK |
|---|---|
| Nucleotide binding | Binds ATP analog (AMPPNP) without Mg²⁺, coordinated via backbone and side chains. |
| Mg²⁺ effect | Inhibits ATP binding and autophosphorylation, likely due to lack of canonical DFG motif. |
| AMP binding | Initially co-purifies with 3’-AMP, displaced by ATP analogs during soaking. |
| Substrate orientation | Peptides align over activation segment, γ-phosphate of ATP positioned toward substrate. |
Mg²⁺ independent ATP binding and catalysis define CASK as a functional but unconventional kinase.
Figure. Phospho-accepting substrate must be bond to catalytic domain in the proper orientation, 4 essential AA in the substrate-binding lobe: 1) Glu (E), Phe (F), Glu(e), Pro (P) (Click to enlarge)
4. Autophosphorylation & Substrate Phosphorylation
- Autophosphorylation:
- Occurs efficiently (~13%) with or without Mg²⁺.
- Identified site: surface loop before GFG motif, suggesting trans-autophosphorylation.
- Substrate phosphorylation:
- Neurexin-1, a synaptic PDZ-domain interactor, is phosphorylated by CASK.
- Mg²⁺ effect on catalysis:
- Inhibits autophosphorylation at low concentrations, plateauing at higher levels, with residual phosphorylation (~33%).
⚙️ CASK performs scaffold-recruited phosphorylation, integrating kinase activity into MAGUK scaffolding roles.
5. Key Structural and Biochemical Insights
| Feature | CASK vs Conventional Kinases |
|---|---|
| DFG motif | GFG (Asp→Gly), prevents Mg²⁺ coordination. |
| Catalytic loop Asn | Replaced by Cys, alternative stabilization of Asp141. |
| Mg²⁺ dependence | Mg²⁺ independent, Mg²⁺ is inhibitory. |
| Activation segment | Ordered, ready for substrate binding (unlike many kinases). |
| Autophosphorylation | Trans, distant from catalytic cleft, surface loop site. |
| Substrate recruitment | Via PDZ-mediated scaffold (e.g., neurexin-1). |
6. Biological Relevance and Broader Implications
- Brain: CASK abundant, where Mg²⁺ and ATP are relatively low, favoring its unique catalytic activity.
- Synaptic function: Combines MAGUK scaffold with neurexin phosphorylation, regulating synapse formation and function.
- Pseudokinase re-evaluation: 10% of kinome thought to be pseudokinases—CASK suggests many could retain cryptic catalytic function.
7. Interesting Highlights & Inspiration
- “Active pseudokinase”: Redefines CASK and perhaps other pseudokinases.
- Unusual catalytic mechanism:
- DFG to GFG swap allows activity without Mg²⁺.
- Cys146 compensates for missing Asn—novel stabilization.
- Dual role scaffold-kinase: MAGUK’s integration of structure and function.
- Synaptic phospho-regulation of neurexin via scaffolded kinase.
- Low Mg²⁺ in neurons might enable CASK activity, with potential to modulate under pathophysiological conditions.
RD’s Reflections
- Beautiful example of molecular evolution: “pseudokinase” CASK reactivated with a unique mechanism.
- Synapse-specific kinase activity scaffolded by MAGUK — potential for modular synaptic signaling hubs.
- Raises fundamental questions about other pseudokinases — how many are actually active under specific contexts?
- Mechanistically fascinating: a kinase that evolved to avoid Mg²⁺ dependence — very different from the classic model.
