Cdk5 is regulated by two central mechanisms, namely protein-protein interaction and phosphorylation (Lim et al. 2003
). A majority of the Cdk5-interacting proteins that have been identified interact with Cdk5 through p35, while a small group of proteins interact directly with Cdk5 (Lim et al. 2003
). For instance, casein kinase 2 inhibits Cdk5 activity by inhibiting Cdk5/p35 complex formation by binding to both Cdk5 and p35 independently (Lim et al. 2004
). To our knowledge, this is the first report that a cytoskeletal protein (G-actin) binds to Cdk5 and inhibits its kinase activity directly, without disrupting Cdk5/p35 or Cdk5/p25 complex formation.
F-actin specifically interacts with the p10 region of p35 in an actinin-independent manner, presumably through interactions with the enriched basic residues found in the p10 sequence (Hou et al. 2007
). Our study suggests that the binding of F-actin to p35 does not hinder Cdk5/p35 complex formation or inhibit its activity, because Cdk5 kinase binding and activating domains do not reside in the p10 region (Poon et al. 1997
). F-actin also directly binds to Cdk5/p35 complexes in our study, suggesting that F-actin may serve as an anchoring cytoskeleton for Cdk5/p35 complexes in the neuronal periphery. How F-actin modestly interacts with Cdk5/p25 complexes as compared to Cdk5/p35 is unknown, since neither Cdk5 nor p25 interacts with F-actin. Perhaps Cdk5 partially stabilizes a weak association of p25 with F-actin, or the association of Cdk5 with p25 may create a novel binding pocket for F-actin.
It has been suggested that p25 has more potency than p35 to activate Cdk5 due to its longer (5~10- fold) half-life in cells (Patrick et al. 1999
). However, to the best of our knowledge, this is the first report that, when Cdk5 complexes were prepared from individually purified Cdk5, p35 or p25, Cdk5/p25 complexes had higher catalytic activity compared with Cdk5/p35 complexes. Cdk5/p25 complexes indeed showed a greater rate of phosphorylation of HH1 than Cdk5/p35 complexes. This implies that there are differences in the conformation of the Cdk5 molecule between the two complexes. Perhaps the substrate-binding site is more accessible in the Cdk5/p25 complex than in the Cdk5/p35 complex, allowing for faster turnover. Hashiguchi et al showed that recombinant co-expressed complexes of Cdk5/p25-His6
have significantly higher intrinsic activity (6-fold) on tau and HH1 than Cdk5/p35-His6
complexes (Hashiguchi et al. 2002
). However, a recent study reported that there is no difference in kinetics of tau or histone phosphorylation by co-expressed complexes of His6
-Cdk5/GST-p35 versus His6
-Cdk5/GST-p25(Peterson et al. 2010
). These contradicting results might be attributed to differences in experimental paradigm, i.e, Cdk5 complexes purified from cells with co-expressed Cdk5 and its activators may include additional interacting molecules.
The modest inhibitory effects of Cdk5/p25 activity observed in in vitro kinase assays with F-actin are most likely due to the presence of residual G-actin after monomer-polymer equilibrium and its interaction with Cdk5/p25 complexes. The reason why we see more inhibition of Cdk5/p25 as compared to Cdk5/p35 is unknown. Considering the significantly higher amount of Cdk5/p25 complexes in the supernatant as compared to Cdk5/p35 after co-sedimentation with F-actin, it is possible that F-actin-free Cdk5 complexes are more susceptible to G-actin inhibition. Since G-actin can directly bind to Cdk5, one of the potential mechanisms is that F-actin might hinder the G-actin binding of Cdk5 and thus may protect Cdk5 complex activity. Alternatively, F-actin may efficiently incorporate G-actin to the F-actin polymer and thus dissociate it from Cdk5.
We have shown that G-actin is a potent inhibitor of the kinase activity, with an IC50
value of about 37 nM. This potency suggests that G-actin can suppress Cdk5 activity in the cytoplasm, especially in actin-rich regions, such as neuronal periphery, dendritic spine, and growth cone. Thus, our study suggests that G-actin may play a key role in Cdk5 activity in these regions. Indeed we observed co-localization of Cdk5 with both G-actin and F-actin in neuronal soma and neurites by confocal microscopy (data not shown). The functional implications for a differential effect of F-actin versus G-actin on Cdk5 kinase activity are not clear. Within a dendritic spine, there is a stable pool of F-actin in the spine core, while a dynamic pool of F-actin and G-actin rests in the spine shell (Racz & Weinberg 2006
, Honkura et al. 2008
), as well as the growth cone tip of the axon (Dent & Gertler 2003
). Since enhancement of F-actin polymerization leads to a reduction of G-actin, the activity of F-actin-associated Cdk5 complexes would be sustained. This would lead to further actin polymerization in the growth cone tip, where Cdk5 interacts with and phosphorylates a plethora of proteins to regulate cytoskeletal dynamics. These observations are not restricted to purified molecules, since our observations were replicated using native Cdk5 complexes freshly immunoprecipitated from mouse brain. In addition, G-actin inhibits Cdk5 complex activity independent of p35 based on our p35−/−
mouse study. These data also substantiate our point that G-actin inhibits Cdk5 activity through its direct interaction with Cdk5. While it was reported previously that p35−/−
mice showed no Cdk5-associated kinase activity (Chae et al. 1997
), we found that a lower level of Cdk5 kinase activity still exists in anti-Cdk5 immunoprecipitates from p35−/−
mouse brain. Perhaps p39, and recently identified Cyclin I, are involved in the residual Cdk5 activity in the p35−/−
mouse brain. In addition, p39 was also associated with the actin cytoskeleton, suggesting similar regulation by actin (Humbert et al. 2000
It has been reported that p35 binds directly to α/β-tubulin and microtubules, which block p35 interaction with Cdk5 and therefore inhibit Cdk5/p35 activity (Hou et al. 2007
). In addition, p35 induces microtubule assembly and bundling, modulating microtubule dynamics in vitro
(Hou et al. 2007
). On the other hand, Kaminosono et al.
reported that Cdk5/p35 disrupts microtubule formation, thereby suppressing aggregation of mutant Huntington proteins (Kaminosono et al. 2008
). This discrepancy may be due to differences in experimental paradigm since microtubule stabilization was observed with purified p35 in vitro
, while microtubule-destabilization was observed in cultured cell lines possessing the Cdk5/p35 complex. However, in neuronal cells Cdk5 and p35 immunoreactivity coincides best with peripheral regions rich in actin filaments, such as the leading edges of growth cones, while tubulin immunoreactivity is mostly in the cell soma and axons (Nikolic et al. 1996
). We have shown that p35/p25 colocalized with both F-actin and G-actin in soma and neurites in cultured cortical neurons. These co-localizations were enhanced upon F-actin clumping as a result of cytochalasin D exposure and weakened upon masking of phalloidin-stained F-actin due to competitive binding of jasplakinolide. Nonetheless, the overall cellular distribution of p35/p25 did not alter after toxin treatment, which might be due to the fact that p35 also binds to microtubules (Hou et al. 2007
). Indeed, we found that p35 colocalized with βIII-tubulin better than with F-actin in control neurons (data not shown).
Our model of actin/p35/Cdk5 interaction () will best apply to the regulation of Cdk5 activity in actin-enriched cell compartments, such as growth cone tips and synaptic dendrites. Indeed, Cdk5 and p35 are highly enriched in these areas and promote actin polymerization and neurite outgrowth through regulation of Rac/Pak1 signaling (Xie et al. 2006
, Nikolic et al. 1998
). Cdk5/p35 complexes can anchor to F-actin via direct interaction of p35 with F-actin or through α-actinin, and promote actin polymerization on site (). G-actin is also highly concentrated in this region and inhibits Cdk5 activity if it is not attached to F-actin, leading to spatial restriction of the Cdk5 activity to Cdk5/p35 complexes attached to F-actin. This tight regulation of Cdk5/p35 activity may be important for unidirectional extension of filopodia, and further extension of the F-actin will accumulate Cdk5/p35 complexes along the actin cytoskeleton. Cdk5/p25, on the other hand, lacks an association with F-actin. Thus its activity is not tightly regulated along F-actin, leading to its activation in the rest of the cytoplasm. F-actin polymerization is also essential for synaptic hypertrophy (or maturation), and Cdk5/p35 activity will also be tightly regulated along F-actin in dendrites, while G-actin will inhibit Cdk5 complexes not attached to actin filaments.
Schematic diagram of actin/p35/Cdk5 interaction and regulation of Cdk5 activity in an extending growth cone tip.
Abnormalities in cytoskeletal organization and/or dynamics are a common feature of many neurodegenerative disorders, including AD (Mendoza-Naranjo et al. 2007
). Pathological actin in the form of F-actin has been found throughout Hirano bodies, which are cytoplasmic inclusions found in several neurodegenerative diseases (Galloway et al. 1987
). Actin is also a component of cofilin-actin rods, inclusion-like structures described in hippocampal and cortical neurons of post-mortem AD brains (Minamide et al. 2000
). However, how actin cytoskeletal alterations lead to neurodegeneration is unclear (Minamide et al. 2000
). Intriguingly, tau-induced neurodegeneration is associated with the accumulation of F-actin and the formation of actin-rich rods in Drosophila and mouse models of tauopathy. Additionally, human amyloid-β peptide synergistically enhances the ability of wild-type tau to promote alterations in the actin cytoskeleton and mediate neurodegeneration (Fulga et al. 2007
). In light of our novel discovery of the actin cytoskeleton as a modulator of Cdk5, it is possible that chronic activation of Cdk5 leads to abnormal F-actin accumulation in the cell soma and depletion of G-actin. This would result in the loss of G-actin-mediated Cdk5 inhibition and the abnormal activation of Cdk5 in the periphery, leading to synaptic degeneration. Further study will be necessary to address how Cdk5 dysregulation leads to synaptic loss in the context of actin regulation.
In summary, we have determined that G-actin but not F-actin potently suppresses Cdk5/p35 and Cdk5/p25 activities without disrupting the complexes. This effect is not substrate-selective, as G-actin inhibited phosphorylation of both HH1 and tau proteins. G-actin mainly binds to Cdk5 and p35 but not p25, whereas F-actin binds to p35 but not Cdk5 or p25. G-actin-mediated inhibition of Cdk5 complexes is not dependent on p35, suggesting its inhibition is through its direct interaction with Cdk5. Finally, actinin had no effect on actin-Cdk5 complex interaction or G-actin-mediated inhibition of Cdk5 complex activity. This actin-mediated regulation of Cdk5 complex activity will be relevant to understanding neuronal development, synaptogenesis, and neurodegenerative disorders where Cdk5 plays a key role in their mechanisms.