A GIT1/PIX/Rac/PAK Signaling Module Regulates Spine Morphogenesis and Synapse Formation through MLC

Abstract

Three of seven recently identified genes mutated in nonsyndromic mental retardation are involved in Rho family signaling. Two of the gene products, ␣-p-21-activated kinase (PAK) interacting exchange factor (␣PIX) 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 ␣PIX and PAK3 mutations affect cognitive functions in mental retardation.

Rho-linked genes and neurological disorders

“…GIT1 was shown to target ßPix to the synapse, where it locally activates Rac1 and PAK. Importantly, disruption of the synaptic localization of GIT1 or interfering with ßPix function, perturbs spine morphogenesis and synapse formation [91]. Consistent with these findings, the Drosophila homologue, dPIX, has been demonstrated to play a major role in regulating post-synaptic structures and protein localization at the glutamatergic neuromuscular junction [64].…”

“…They noticed that these defects were associated with reduced expression of AMPARs at the synapse and LTP [9]. In a parallel study, Zhang et al demonstrated that PAK1 and 3 regulate spine morphogenesis by triggering the phosphorylation of MLC, the latter resulting in an increase in dendritic spine size and synapse formation [91]. More recently, mice lacking the PAK3 gene were generated, and analysis of these mice showed selective impairment in late-phase hippocampal LTP, a distinct form of long-term synaptic plasticity involving new gene expression.…”

Regulation of actin cytoskeleton dynamics by Arf-family GTPases

“…GIT1 has also been shown to modulate synapse formation in hippocampal neurons, through recruitment of the GIT-PIX-PAK complex to dendritic spines and synapses [74]. However, recruitment of GIT1 to spines does not require interaction with paxillin.…”

Alteration of Synaptic Network Dynamics by the Intellectual Disability Protein PAK3

“…There is also strong evidence for a redistribution of polyribosomes from dendritic shafts to active synapses (Ostroff et al, 2002). Activity might thus promote local movement of a number of molecules into spines, and PAK3 might not only be part of these changes but also participate in their regulation, notably through its possible action on proteins, such as cofilin, myosin II regulatory light chain, or cortactin (Edwards et al, 1999;Hashimoto et al, 2001;Zhang et al, 2005).…”