Functional analyses of human and zebrafish 18-amino acid in-frame deletion pave the way for domain mapping of the cerebral cavernous malformation 3 protein

Voss, K., Stahl, S., Hogan, B.M., Reinders, J., Schleider, E., Schulte-Merker, S., and Felbor, U.
Human Mutation   30(6): 1003-1011 (Journal)
Registered Authors
Hogan, Ben M., Schulte-Merker, Stefan
cerebral cavernous malformation, CCM3, PDCD10, CCM2, STK25, MST4
MeSH Terms
  • Amino Acids/genetics*
  • Animals
  • Apoptosis Regulatory Proteins/chemistry
  • Apoptosis Regulatory Proteins/genetics*
  • Apoptosis Regulatory Proteins/metabolism
  • Cardiovascular Abnormalities/embryology
  • Cardiovascular Abnormalities/metabolism
  • Carrier Proteins/metabolism
  • Cell Line
  • Exons/genetics
  • Frameshift Mutation/genetics*
  • Gene Knockdown Techniques
  • Heart/embryology
  • Humans
  • Intracellular Signaling Peptides and Proteins/metabolism
  • Membrane Proteins/chemistry
  • Membrane Proteins/genetics*
  • Membrane Proteins/metabolism
  • Microtubule-Associated Proteins/metabolism
  • Phenotype
  • Phosphorylation
  • Phosphoserine/metabolism
  • Phosphothreonine/metabolism
  • Protein Binding
  • Protein Interaction Mapping*
  • Protein Serine-Threonine Kinases/metabolism
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins/chemistry
  • Proto-Oncogene Proteins/genetics*
  • Proto-Oncogene Proteins/metabolism
  • Sequence Deletion*
  • Zebrafish/embryology
  • Zebrafish/genetics*
  • Zebrafish Proteins/chemistry
  • Zebrafish Proteins/genetics*
19370760 Full text @ Hum. Mutat.
Cerebral cavernous malformations (CCMs) may cause recurrent headaches, seizures, and hemorrhagic stroke and have been associated with loss-of-function mutations in CCM1/KRIT1, CCM2, and CCM3/programmed cell death 10 (PDCD10). The CCM3/PDCD10 amino acid sequence does not reveal significant homologies to protein domains with known structure. With the help of the only published human in-frame deletion of the CCM3 gene (c.97-?_150+?del), CCM3:p.L33_K50del, we have identified the interaction domain of CCM3 with the oxidant stress response serine/threonine kinase 25 (STK25, YSK1, SOK1) and with the mammalian Ste20-like kinase 4 (MST4, MASK). Consistently, nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analyses revealed two STK25 phosphorylation sites at serine 39 and threonine 43. The corresponding in-frame deletion of zebrafish ccm3a, dccm3:p.L31_K48del, also resulted in impaired interaction with STK25 and MST4. In agreement with the observed redundant biochemical functionality of zebrafish ccm3a and its duplicate ccm3b, simultaneous inactivation of both genes resulted in a progressive cardiovascular phenotype in zebrafish indistinguishable from ccm1 and ccm2 mutants. The pronounced cardiovascular dilatations could be recapitulated by morpholino-induced in-frame skipping of the exon encoding the STK25 and MST4 binding site of zebrafish Ccm3a if Ccm3b was repressed in parallel. Using a novel zebrafish model of CCM, we could thus demonstrate that the newly mapped STK25 and MST4 interaction domain within the CCM3 protein plays a crucial role for vascular development in zebrafish.
Genes / Markers
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Engineered Foreign Genes
Errata and Notes