A GIT1/PIX/Rac/PAK signaling module regulates spine morphogenesis and synapse formation through MLC.
Three of seven recently identified genes mutated in nonsyndromic mental retardation are involved in Rho family signaling. Two of the gene products, alpha-p-21-activated kinase (PAK) interacting exchange factor (alphaPIX) and PAK3, form a complex with the synaptic adaptor protein G-protein-coupled receptor kinase-interacting protein 1 (GIT1). Using an RNA interference approach, we show that GIT1 is critical for spine and synapse formation. We also show that Rac is locally activated in dendritic spines using fluorescence resonance energy transfer. This local activation of Rac is regulated by PIX, a Rac guanine nucleotide exchange factor. PAK1 and PAK3 serve as downstream effectors of Rac in regulating spine and synapse formation. Active PAK promotes the formation of spines and dendritic protrusions, which correlates with an increase in the number of excitatory synapses. These effects are dependent on the kinase activity of PAK, and PAK functions through phosphorylating myosin II regulatory light chain (MLC). Activated MLC causes an increase in dendritic spine and synapse formation, whereas inhibiting myosin ATPase activity results in decreased spine and synapse formation. Finally, both activated PAK and activated MLC can rescue the defects of GIT1 knockdown, suggesting that PAK and MLC are downstream of GIT1 in regulating spine and synapse formation. Our results point to a signaling complex, consisting of GIT1, PIX, Rac, and PAK, that plays an essential role in the regulation of dendritic spine and synapse formation and provides a potential mechanism by which alphaPIX and PAK3 mutations affect cognitive functions in mental retardation.
Zhang
Huaye
H
Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA.
Webb
Donna J
DJ
Asmussen
Hannelore
H
Niu
Shuang
S
Horwitz
Alan F
AF
D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D049229:Dendritic Spines / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D004075:Diglycerides / Q000378:metabolism; D000076122:Disks Large Homolog 4 Protein; D004622:Embryo, Mammalian; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D015870:Gene Expression / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology; D007150:Immunohistochemistry / Q000379:methods; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009024:Morphogenesis / Q000187:drug effects / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism*; D009474:Neurons / Q000166:cytology; D010750:Phosphoproteins / Q000172:deficiency / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D064067:Rho Guanine Nucleotide Exchange Factors; D013569:Synapses / Q000187:drug effects / Q000502:physiology*; D014162:Transfection / Q000379:methods; D054462:p21-Activated Kinases
D018797:Cell Cycle Proteins; D004075:Diglycerides; D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; C485648:Git1 protein, rat; D020662:Guanine Nucleotide Exchange Factors; D006576:Heterocyclic Compounds, 4 or More Rings; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D018994:Myosin Light Chains; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D064067:Rho Guanine Nucleotide Exchange Factors; D049452:Green Fluorescent Proteins; C472645:blebbistatin; C017923:rac-1-oleyl-2-4-(3-pyrenyl)butanoylglycerol; C515910:Pak2 protein, rat; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases