To investigate potential phosphorylation by Cdk5, CASK was divided into different domains that were expressed as GST fusion proteins and used as substrates in an in vitro
kinase assay with purified active Cdk5. The autoradiography results show that the L27, CaMK and GUK domains were phosphorylated by Cdk5 to a similar extent as the known substrate Ndel1 (also known as Nudel) (). Next, to determine the sites, we used two-dimensional phosphoaminoacid analysis to distinguish phospho-serine, -threonine and –tyrosine residues. The results suggested that serine residues are the major sites of phosphorylation in the L27 and CaMK domains (), and that threonine residues are primarily phosphorylated in the GUK domain (data not shown). The L27 and CaMK each contain only one serine that can be phosphorylated by Cdk5, indicating that the major sites are Ser 395 and Ser 51 respectively. Likewise, the GUK domain contains only one proline-directed threonine residue, Thr 846. We also processed samples for mass spectrometry and with a combination of data from a cellular system and mouse brain synaptosomes, we were able to detect phosphorylation of CASK at both Ser 51 and Ser 395 (Supplemental Figure 1
We next tested for an in vivo
association between the Cdk5 activator p35 and CASK in brain lysate. Some Cdk5 substrates, such as Amphiphysin-1 (Floyd et al., 2001
), bind p35. Immunoprecipitates made with CASK antibodies demonstrated an interaction with p35 and Cdk5 (). Likewise, p35 immunoprecipitates from wild-type mouse brains contained endogenous CASK (). While the total amount of CASK interacting with p35 in this snapshot is small, it is consistent with the transient nature of a substrate-kinase relationship. Also, CASK was not immunoprecipitated from littermate p35 deficient mice or with a control antibody, indicating specificity to the association between CASK and p35.
To analyze phosphorylation of CASK in vivo
, we made phosphorylation state-specific antibodies. Phospho-Ser 51 (pS51) and phospho-Ser 395 (pS395) antibodies recognized several bands in brain lysate but only one in samples enriched for CASK by immunoprecipitation (; Supplemental Figure 2
), suggesting that both phospho-antibodies recognize a form of CASK present in embryonic mouse brains. Subsequently, CASK immunoprecipitates were made from lysates of Cdk5 deficient or littermate brains and probed for phospho-CASK. While total CASK did not differ between wild-type and Cdk5 knockout mice, pS395 and pS51 levels were markedly decreased in the absence of Cdk5 (). We were also able to detect decreased phosphorylation of CASK by other Cdk5 loss-of-function methods including dominant negative and pharmacological approaches (Supplemental Figure 3
). Phosphorylation of T846 did not appear to be a major event in vivo
(data not shown). These data indicate that Cdk5 is a major kinase for phosphorylation of S51 and S395 in vivo
We next examined the temporal profile of CASK phosphorylation in the developing brain. Interestingly, phosphorylation increases slightly during postnatal development and plateaus around P4 while the total amount of CASK remains the same (). Phosphorylation of Cdk5 substrates important for the neuronal migration program, such as FAK, decrease in the postnatal period (Xie et al., 2003
). However, this profile suggests a more important role for Cdk5-dependent phosphorylation of CASK during later stages of brain development and the more mature nervous system.
To understand a potential role for CASK phosphorylation, we examined the subcellular distribution of CASK in Cdk5 deficient mice. We used a series of centrifugations to resolve nuclear/mitochondrial, membrane-associated and cytosolic pools of cellular proteins. In wild-type embryonic brains, the highest level of CASK is in the membrane-associated fraction, with a significant amount also in the cytosol (soluble) (). In Cdk5 deficient brains, however, CASK is significantly reduced in the membrane-associated fraction () (44.6±2.6% vs 24.4±2.8% of total CASK is membrane-associated; Mean±SEM; control vs KO/mutant), while the soluble pool of CASK is increased (34.3±2.5% vs 49.7±2.9% is soluble). In comparison, the NR2A subunit of NMDA receptors, another Cdk5 substrate, is not altered when comparing control and Cdk5 deficient membrane fractions (). These data indicate Cdk5 activity is necessary for the appropriate membrane localization of CASK. This is an intriguing result as CASK is a member of the MAGUK family, proteins that play significant intracellular scaffolding roles at cellular membranes.
Cdk5-dependent phosphorylation mediates membrane association of CASK
We next determined if elimination of CASK phosphorylation by Cdk5 was directly responsible for this distribution phenotype. To this end, CASK constructs tagged with myc and mutated from serine to alanine at S51, S395, or both, were created. Neuroblastoma CAD cells were cotransfected with the different constructs and active Cdk5, then differentiated and fractionated. Cells expressing wild-type CASK-myc or the single site alanine mutants had a CASK distribution similar to control brains. In cells expressing the double alanine mutant S51/395A, however, the transfected CASK was depleted from the membrane-associated fraction () (58.5±3.2% vs 18.7±2.4% is membrane-associated) and increased in the cytosol (25.8±2.4% vs 43.8±2.6% is soluble), recapitulating the localization phenotype seen in the Cdk5 deficient mice. While similar, this shift in the localization of CASK is even stronger than that seen in the Cdk5 knockout mice, likely due to the residual phosphorylation that remains in the mouse and that the stoichiometry of phosphorylation may be higher in CAD cells. These observations argue that loss of Cdk5-dependent phosphorylation at both S51 and S395 is necessary for removal of CASK from the membrane-associated fraction.
To gain an understanding of the subcellular localization of endogenous phospho-CASK in neurons, we used the pS395-CASK antibody to probe fractions from wild-type embryonic mice brains. Since total CASK is enriched in membrane-associated compartments, an effort was made to equalize all of the fractions prior to immunoprecipitation in order to get a reasonable determination of where phosphorylation was occurring. The results suggest phospho-CASK is enriched in membrane fractions with a significant amount also present in the soluble pool (). This result provides in vivo evidence that endogenous phospho-CASK is present at membranes and supports our data using overexpression of the double alanine mutant in CAD cells and neurons.
Having seen a decrease in phosphorylation and a resulting redistribution of CASK in Cdk5 loss-of-function mice, we next tested if CASK was altered in a Cdk5 gain-of-function model. We employed a bitransgenic mouse model using the tetracycline-controlled transactivator (tTA) system to drive inducible expression of the Cdk5 activator p25 under control of the CaMKII promoter (CK-p25 mice) (Cruz et al., 2003
). Bitransgenic mice were raised in the presence of doxycycline for 4–6 weeks postnatal before induction of p25. We then examined CASK phosphorylation and subcellular distribution in CK-p25 mice where the p25 transgene has been expressed for only two weeks. At this timepoint, similar to other Cdk5 substrates such as Pak1 and NR2A (Fischer et al., 2005
), CASK phosphorylation was increased in brains from CK-p25 mice (). Furthermore, while in Cdk5-deficient brains CASK was depleted from membranes, in p25-overexpressing brains CASK is more enriched in membrane fractions compared to wild-type littermates () (48.6±1.9% vs 66.0±2.7% is membrane-associated). Also, the cytosolic pool of CASK is depleted in p25 transgenic mice (34.1±2.6% vs 19.2±2.4% is soluble) and NR2A is unchanged in p25 fractions (). Taken together, these results confirm that Cdk5-dependent phosphorylation of CASK regulates the subcellular distribution of CASK in a dynamic fashion. As phospho-CASK levels increase, CASK shifts from the cytosolic pool of proteins to a membrane-associated pool.
While this data suggests that Cdk5 activity can directly regulate CASK distribution to membrane-associated pools, we sought to determine what effect Cdk5 activity has on CASK distribution specifically to synaptic membrane-associated pools. To this end, we performed synaptosome preparations using wild-type adult mice brains to determine the distribution of CASK, and more importantly, phospho-CASK. As expected, a pool of CASK was distributed to LP1, the presumptive synaptosomal membrane fraction (). We next performed CASK immunoprecipitations from H (the homogenate or input fraction), LS1 (the synaptosome cytosol) and LP1. Interestingly, we found that phospho-CASK is relatively more enriched in synaptosomal membrane fractions than total CASK (). Finally, we determined if Cdk5 loss-of-function altered CASK distribution to synaptic membranes. Rather than using the Cdk5 deficient mice, which die around birth, we crossed floxed Cdk5 and αCaMKII-Cre (Yu et al., 2001
) mice to generate forebrain-specific Cdk5 conditional knockout (Cdk5 cKO) mice. Importantly, CASK phosphorylation was markedly reduced in the Cdk5 cKO mice relative to control littermates (). Preparation of synaptosomes from Cdk5 cKO mouse brains revealed a strikingly altered distribution of CASK relative to control (). In synaptosomes from Cdk5 cKO mice, CASK is significantly decreased in the LP1 membrane fraction and increased in the LS1 and LS2 cytosolic fractions relative to control littermates. Importantly, synaptophysin, a synaptic vesicle associated protein, is not altered (). This data suggests that Cdk5-dependent phosphorylation in part mediates CASK distribution to synaptic membranes.
Cdk5 mediates CASK distribution in synaptosomes
To determine if Cdk5-mediated phosphorylation of CASK, and CASK distribution to synaptic membrane-associated pools, is important during synaptogenesis, we used a synapse formation assay where heterologous cells (such as 293) overexpressing a synaptic cell adhesion molecule (such as Neuroligins or SynCAM) are cocultured with pontine explants from late embryonic/early postnatal mouse brains. After a few days, axonal processes emanating from the pontine explants are capable of forming presumptive presynaptic terminals enriched with CASK and presynaptic markers at points of contact with the heterologous cells expressing the synaptic cell adhesion molecules (Scheiffele et al., 2000
). We cocultured pontine explants from wild-type or Cdk5-deficient mice with 293 cells expressing SynCAM or Neuroligins. There was no significant difference in the number or length of processes emanating from Cdk5-deficient explants relative to wild-type (Supplemental Figure 4
). At low magnification, it is apparent that Cdk5 deficient explants display less clustering of CASK on the 293 cells, which are visualized with Hoechst (). At higher magnification, where the 293 cells are visualized by cotransfected GFP, quantification of the number of puncta per surface area of 293 cell (Biederer and Scheiffele, 2007
) demonstrates significantly less clustering of CASK, suggesting that Cdk5-mediated phosphorylation is important for CASK recruitment to developing synapses (). In addition, Cdk5-deficient processes demonstrated a mild but significant decrease in the amount of synaptophysin clusters (, Supplemental Figure 5
), suggesting that Cdk5 is important for synapse formation.
Cdk5 is required for CASK recruitment to presynaptic terminals and for synapse formation
We also used a second assay of in vitro
synapse formation to complement the pontine explant experiments. In this assay, cortical neurons were cultured from wild-type or Cdk5-deficient littermates with 293 cells that had been transfected with SynCAM and GFP. In wild-type cocultures, CASK and the presynaptic marker Bassoon clustered at sites of contact with the 293 cells (). However, in Cdk5-deficient cocultures, CASK was often absent at sites of contact where Bassoon accumulated (). Quantification, determined by the fractional area of staining per surface area of 293 cell (Biederer and Scheiffele, 2007
), indicated a marked decrease in CASK at developing synapses made by Cdk5-deficient neurons (). There was also a more mild, but significant, decrease in the percentage of 293 cells displaying clusters of Bassoon, once again indicating that Cdk5 activity is important for synapse formation ().
Finally, we infected explants made from wild-type embryonic mice with herpes simplex virus encoding GFP-tagged wild-type or S51/395A CASK and investigated the clusters that formed on 293 cells (). Although these clusters were larger than seen with endogenous CASK (likely due to the overexpression system), comparison of their size with the scale bar indicates they are puncta. Quantification determined that there were significantly fewer GFP-S51/395A CASK clusters (), suggesting that mutation of Ser 51 and Ser 395 limits the ability of CASK to cluster at developing synapses.
Given that membrane localization and recruitment of CASK to developing synapses is Cdk5-dependent led us to hypothesize that phosphorylation may promote the interaction between CASK and proteins enriched at synaptic membranes. CASK was originally identified as an intracellular interactor of neurexin proteins (Hata et al., 1996
), which are the presynaptic partners mediating neuroligin-induced synaptogenesis (Dean et al., 2003
; Nguyen and Sudhof, 1997
; Scheiffele et al., 2000
). Interestingly, CASK association with neurexins is significantly decreased in membrane fractions from Cdk5 deficient mice compared to wild-type mice (), suggesting that CASK interaction with synaptic cell adhesion molecules is also Cdk5-dependent.
CASK interactions with presynaptic proteins are regulated by Cdk5
We next examined the interaction of CASK with Mint1 and Veli. The tripartite complex of CASK, Mint1 and Veli is established in mammalian brains (Butz et al., 1998
; Olsen et al., 2005
; Olsen et al., 2006
), and is evolutionarily conserved and well understood in organisms such as C.elegans
(Kaech et al., 1998
; Simske et al., 1996
). Triple knockout mice of all Veli (also known as MALS) isoforms have decreased CASK levels and reduced EPSCs relative to wild-type mice (Olsen et al., 2005
; Olsen et al., 2006
). Mint1 binds the essential synaptic vesicle exocytosis protein Munc18-1 (Okamoto and Sudhof, 1997
), and Mint1 deficient mice have impairments in GABAergic transmission (Ho et al., 2003
). As expected, immunoprecipitates of Veli from wild-type mouse brain membranes demonstrate a strong interaction with CASK. However, much less CASK associated with Veli in the absence of Cdk5 activity (). Similarly, Mint1 immunoprecipitates made from wild-type mice membrane fractions contained much more CASK than those made from Cdk5 deficient mice ().
We next examined the interaction of CASK with α1B subunits of N-type calcium channels. This interaction has been demonstrated with GST-pulldowns (the C-terminal tail of α1B interacts with the SH3 domain of CASK in vitro
) (Maximov and Bezprozvanny, 2002
; Maximov et al., 1999
) and immunoprecipitation from chick brain lysate (Khanna et al., 2006
). Indeed, endogenous α1B from embryonic wild-type brain membranes coimmunoprecipitated CASK. Intriguingly, endogenous α1B immunoprecipitates made from brain membranes of Cdk5 deficient littermates did not contain CASK (). This result suggests that CASK interaction with N-type calcium channels is abolished in vivo
in the absence of Cdk5 activity.
Taken together, our data suggests that in the absence of Cdk5-mediated phosphorylation of CASK at Serine 51 and Serine 395, CASK is depleted from neuronal membranes, is not recruited to developing synapses, and has a decreased association with presynaptic machinery, such as N-type voltage gated calcium channels, Veli, Mint1 and neurexins. While CASK displays decreased phosphorylation and depletion from membranes in Cdk5-deficient mice, CASK phosphorylation and localization to membranes is increased in Cdk5 gain-of-function (CK-p25) mice. Therefore, we prepared endogenous N-type calcium channel immunoprecipitates from brain membranes of CK-p25 mice and found that the in vivo interaction between the α1B subunit and CASK is increased relative to wild-type littermates (). This data confirms that Cdk5 promotes the interaction between CASK and N-type calcium channels.
While our data suggests that CASK recruitment to presynaptic membranes and subsequent interactions with essential presynaptic machinery are Cdk5-dependent, we wanted to understand what impact altering these interactions might have in neurons. To this end, wild-type or double alanine mutant CASK was introduced to primary hippocampal neurons using high efficiency electroporation and potential changes in [Ca2+
in response to high K+
stimulation was assessed. After 10–14 days in culture, ratiometric calcium imaging was performed using the conventional indicator fura-2 AM. Upon depolarization with high K+
, calcium influx into untransfected or wild-type CASK transfected neurons was very prominent (). However, in S51/395A CASK transfected neurons, the peak change in [Ca2+
was moderately decreased (). We also found that Cdk5 knockdown, mediated by two distinct RNAi constructs, caused a significant decrease in the peak change in [Ca2+
(Supplemental Figure 6
). This data demonstrates that calcium influx into neurons expressing a mutant form of CASK that cannot be phosphorylated by Cdk5 is significantly compromised.
CASK is important for depolarization-dependent calcium influx in hippocampal neurons
It has been suggested that CASK interaction with calcium channels is limited to Cav
2 channels (Spafford and Zamponi, 2003
), so to determine the source of calcium influx primarily affected by double alanine mutant CASK we pretreated transfected hippocampal neurons with ω-conotoxin GVIA and ω-agatoxin IVA, inhibitors of N- and P/Q-type channels respectively. While calcium influx was significantly decreased with the treatment, upon high K+
-mediated depolarization a prominent amount was still detectable, consistent with the fact that there are many other sources of calcium influx in neurons. We found that pretreatment with blockers of N- and P/Q-type calcium channels eliminated the significant difference in calcium influx between wild-type and S51/395A-CASK transfected neurons (). Pretreatment with APV, which blocks NMDAR-mediated calcium influx, did not eliminate the difference (Supplemental Figure 6
). This data suggests that Cdk5-phosphorylated CASK promotes calcium influx primarily through Cav
2 calcium channels.
When CASK is not phosphorylated, it does not interact with calcium channels embedded in membranes. Therefore, one way to explain the calcium influx phenotype is that eradication of the interaction with N-type channels is akin to CASK loss-of-function. Therefore, to test our hypothesis that regulation of presynaptic calcium channels is an in vivo function of CASK in neurons, we developed a RNAi construct that knocks down CASK levels (). Furthermore, cells cotransfected with CASK RNAi and CASKrescue, a construct containing a silent mutation in the coding sequence within the region targeted by the CASK RNAi, are able to maintain expression of CASK. While calcium influx was not altered when comparing neurons expressing CASK, Ndel1 (Nudel) RNAi or CASK RNAi in conjunction with CASKrescue, the change in [Ca2+]i was significantly decreased in cells expressing CASK RNAi alone relative to the other conditions (). This data suggests that CASK loss-of-function results in a similar decrease in calcium influx as overexpression of the nonphosphorylatable form of CASK, and that CASK is capable of promoting calcium influx in response to depolarization.
We next sought a more direct means to test the effect of CASK on calcium channels. To this end, calcium currents were recorded through N-type channels (rat α1b, β3, α2δ) stably expressed in TSA cells (Lin et al., 2004
) in the presence or absence of CASK. shows exemplar calcium current records evoked with the indicated voltage-pulse paradigm. Intriguingly, CASK causes an amplifying effect. Next we determined the peak current density (Jpeak
) by measuring peak calcium current as shown (Ipeak
) and dividing by cell capacitance. Population averages are shown in , plotting Jpeak
as a function of test-pulse potential (10 mV for the exemplars in ). These average data confirm that CASK produces a 2–3 fold enhancement of N-type currents, fitting nicely with the calcium imaging. We also utilized G-Q
analysis (Agler et al., 2005
) and determined that an increase of channel open probability appears to account for much of the current augmentation by CASK (Supplemental Figure 7
). Taken in conjunction with the calcium imaging, this data indicates that CASK is capable of modulating the function of N-type channels.
While Cdk5-dependent phosphorylation regulates the subcellular distribution of CASK, and in turn modulates interactions of CASK with presynaptic proteins, we wanted to gain a better understanding of a direct mechanism. Ser 51 and Ser 395 are located in the CaMK and L27 domains of CASK, respectively. Interestingly, the interaction between CASK and liprin-α proteins is dependent on both domains being intact (Olsen et al., 2005
). As liprin-α proteins organize the presynaptic active zone, we tested if Cdk5-dependent phosphorylation of CASK regulated this interaction. Using a GST fusion protein of the liprin-α2 SAM domain, we pulled down overexpressed CASK from transfected 293 cells (). Interestingly, when wild-type CASK, but not S51/395A CASK, was cotransfected with active Cdk5 we noticed a significantly decreased binding between CASK and liprin-α (, lanes 7–8). Other known interactors of CASK, including Mint1, Veli and SAP97, did not display such an altered binding (Supplemental Figure 8
Cdk5 directly regulates the interaction between CASK and liprin-α
We next performed pulldowns from Cdk5-deficient brain lysate. Compared to wild-type, GST-liprin-α pulled down more CASK from Cdk5-deficient brains (), suggesting that Cdk5-dependent phosphorylation of CASK disrupts the association with liprin-α proteins. We next assessed this interaction using lysates from Cdk5 gain-of-function mouse brains. In comparison to wild-type lysate, GST-liprin-α pulled down less CASK from CK-p25 brains (). Taken together, these results suggest that Cdk5-dependent phosphorylation is capable of directly regulating the interaction between CASK and liprin-α proteins, and suggests a potential molecular mechanism of how Cdk5-dependent phosphorylation may regulate CASK subcellular distribution and subsequent interaction with other components of the presynaptic machinery.