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Genes to Cells (2006) 11, 1393-1404. doi:10.1111/j.1365-2443.2006.01027.x
© 2006 Blackwell Publishing or its licensors
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Identifying novel substrates for mouse Cdk5 kinase using the yeast Saccharomyces cerevisiae

Youko Horiuchi1, Akiko Asada2, Shin-ichi Hisanaga2, Akio Toh-e3 and Masafumi Nishizawa1,*

1 Department of Microbiology and Immunology, Keio University School of Medicine, Shinjuku, Tokyo 160-8582, Japan
2 Department of Biological Sciences, Tokyo Metropolitan University Graduate School of Science, Hachioji, Tokyo 192-0397, Japan
3 Department of Biological Sciences, The University of Tokyo Graduate School of Science, Bunkyo, Tokyo 113-0033, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Among the mammalian Cdk family members, Cdk5, activated by the binding of p35, plays an important role in the control of neurogenesis, and its deregulation is thought to be one of the causes of neurodegenerative diseases. Overproduction of Cdk5 and p35 in yeast cells causes growth arrest, probably because of hyperphosphorylation of yeast proteins. We screened mouse brain cDNA that could recover the growth of yeast cells overproducing Cdk5 and p35, hoping that such cDNA encodes a substrate or inhibitor of Cdk5. Mouse brain cDNA library was introduced into a yeast strain in which Cdk5, p35 and mouse cDNA were over-expressed under the control of the GAL promoter, and cDNA plasmids were isolated from the transformants that recovered growth on galactose medium. The analysis of those plasmids revealed that they harbored cDNA that encodes neuronal proteins including SCLIP and CRMP-1, and those with unknown function. We found that Cdk5 could phosphorylate SCLIP and CRMP-1 in vitro and the two proteins in cultured cells showed a mobility shift depending on Cdk5 activity and the presence of specific Ser or Thr residues, indicating that SCLIP and CRMP-1 are likely substrates for Cdk5 in vitro and in cultured cells. Further screening with these systems will enable us to identify more novel substrates and regulators of Cdk5/p35, which will lead to the exploration of Cdk5 function in diverse cellular systems.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Cyclin-dependent kinase (Cdk) plays a key regulatory role in the progression of the cell cycle. Vertebrate cells have various Cdks (Cdk1 to Cdk9) and cyclins (cyclins A, B, C, D, E, H and T), and their different combinations are utilized at different stages of the cell cycle (Morgan 1995). Although being highly homologous to other members of the Cdk family, Cdk5 has not yet been shown to be involved in cell proliferation. Cdk5 is activated by the binding of a neuron-specific activator, p35 or p39 subunit, and the Cdk5-associated kinase activity is predominantly displayed in postmitotic neurons. Cdk5 plays an important role in the control of neurogenesis, including neurite outgrowth, axon guidance, and cell migration (Tsai et al. 1994; Nikolic et al. 1996; Paglini et al. 1998). Cdk5/p35 complex functions in the regulation of actin through phosphorylation of Pak1 (Nikolic et al. 1998), and in synaptic endocytosis through amphiphysin I and dynamin I (Tomizawa et al. 2003). Tau protein is a substrate of Cdk5 and its hyperphosphorylation is believed to be one of the causes of neurodegenerative diseases including Alzheimer's disease (Patrick et al. 1998). Recent reports indicate that Cdk5 plays a regulatory role in the modulation of the activity of various signaling and transcriptional molecules involved in diverse cellular processes not only in neurons but also in various cell types (Li et al. 2004; Rosales et al. 2004). To date, more than 30 substrates have been identified for Cdk5; however, this number of substrates is not sufficient to explain the expanding function of Cdk5 (Lee et al. 2004; Morabito et al. 2004; Moy & Tsai 2004; Tanaka et al. 2004; Xie & Tsai 2004).

In the budding yeast Saccharomyces cerevisiae, Cdc28 cyclin-dependent kinase is responsible for cell cycle transitions (Nasmyth 1993). Pho85 kinase, a member of the yeast Cdk family, has been isolated as the negative regulatory factor of PHO system (Uesono et al. 1987), and is known to associate with ten different cyclin-like partners (Measday et al. 1997). Pho85 has a pleiotropic function including response to nutrient conditions, PLC-pathway, aminoglycoside sensitivity and cell cycle. Mouse Cdk5 and yeast Pho85 share 57% identity in the amino acid sequence and regulation of the two kinases appears similar: Cdk5 and Pho85 do not require phosphorylation of Ser residue in the T-loop for activity unlike Cdks functioning in the cell cycle progression (Poon et al. 1997; Nishizawa et al. 1999b), and phosphorylation of the Tyr residue in the N-terminal region, which is inhibitory to other Cdks, is important for Pho85 and stimulates Cdk5 kinase activities, respectively (Nishizawa et al. 1999b; Zukerberg et al. 2000). In addition, expression of Cdk5 in {Delta}pho85 strain could suppress some of {Delta}pho85 mutant phenotypes by association with cyclins for Pho85 to phosphorylate the substrates of Pho85 including Pho4. Thus, it can be concluded that mouse Cdk5 is a functional homolog of yeast Pho85 kinase (Huang et al. 1999; Nishizawa et al. 1999a). The functional similarities between Cdk5 and Pho85 were extended by recent studies. The G1-specific forms of Pho85 contribute to actin regulation through an interaction with Rvs167 protein (Lee et al. 1998). Cdk5 can phosphorylate amphiphysin I, one of the two mammalian homologs of Rvs167, which is involved in endocytosis at nerve termini and binds to proteins required for endocytosis such as dynamin and clathrin (Floyd et al. 2001).

The yeast cells expressing both Cdk5 and p35 show morphological abnormalities and that overproduction of the two proteins causes a growth defect, which is more prominent in the absence of PHO85, whereas the cells expressing either Cdk5 or p35 alone have no significant growth defect (Huang et al. 1999). These facts suggest the possibility that Cdk5/p35 complex is much more active in yeast cells than the combination of Cdk5 with Pho85-cyclins. The growth defect of yeast cells overproducing Cdk5/p35 complex was, therefore, probably caused by inappropriate phosphorylation of yeast proteins that are important for growth. From this, we inferred that mouse brain cDNA that could suppress the growth arrest could encode an inhibitor or substrate of Cdk5. The presence of overproduced authentic substrate may reduce the extent of phosphorylation of yeast proteins by Cdk5, resulting in recovery from the growth arrest. By screening a mouse brain cDNA library with this system, we could isolate cDNA clones that could restore yeast growth, and among them, we identified SCLIP and CRMP-1 as novel substrates for Cdk5/p35.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Screening and isolation of cDNA clones that recovered growth of yeast cells overproducing Cdk5 and p35

We constructed MFY188 strain in which Cdk5 and p35 were produced under the control of the GAL promoter, and to this strain introduced mouse brain cDNA expression plasmids of which expression were also under the control of the GAL promoter. Growth arrest of MFY188 cells and successful production of Cdk5 and p35 in SGalRaf medium were confirmed (Fig. 1A and Fig. 1B, lane 1). Colonies of transformants were replica-plated on to the SGalRaf medium, and after 5–7 days, cDNA plasmids were recovered from the colonies that formed on the selective medium. Those plasmids that harbor a sufficient length of cDNA insert (more than c. 500 bp) were selected, and after confirmation of their ability to suppress the growth arrest caused by overproduction of Cdk5/p35, the nucleotide sequence of the cDNA insert was determined, and those having a sufficient length of ORF were then subjected to a BLAST search for homologous sequences in the GENBANK database. A list of typical cDNA clones isolated by this screening is shown in Table 1.


Figure 1
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  		Figure 1  Growth arrest of {Delta}pho85 mutant cells by (A) overproduction of Cdk5/p35 and (B) immunoblot analysis of Cdk5 and p35 in the transformants. (A) MFY188 cells expressing Cdk5 and p35 under the direction of the GAL10 promoter were streaked on SD (Glucose) or SGalRaf (Galactose) medium. The growth of yeast cells on SD and SGalRaf medium were photographed after 2 days at 30 °C and after 5 days at 36.5 °C, respectively. (B) Immunoblot analysis of Cdk5 and p35 in the transformants that could grow on the selective medium. The transformants harboring respective cDNA clone (lane 1, vector; lanes 2 and 3, Skp1; lanes 4 and 5, SCLIP; lanes 6 and 7, CRMP-1; lanes 8 and 9, Bpx; and lanes 10 and 11, uncharacterized cDNA [CO804842]) were grown in SGalRaf medium for 30 h, and cell extracts were prepared and subjected to Western analysis with anti-Cdk5 or anti-p35 antibody as indicated.

 

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  		Table 1  List of isolated cDNA clones
 
To confirm that the growth recovery was not caused simply by degradation of either Cdk5 or p35, the status of Cdk5 and p35 in the transformants that could grow on SGalRaf medium was analyzed by Western blotting. The transformants were grown in the presence of galactose for 30 h, so that Cdk5, p35 and the mouse proteins coded by cDNA were fully produced. The results are shown in Fig. 1B in which selected cDNAs were tested. Since these proteins were all produced under the control of the GAL promoter, the signals of Cdk5 and/or p35 should become weaker than those in the control where only Cdk5 and p35 were produced, if the cDNA product caused degradation of either Cdk5 or p35. The results in Fig. 1B show that the amounts of Cdk5 and p35 in the respective transformants were almost equivalent to those in the control cells, indicating that the cDNA clones tested could recover the growth of yeast cells not by degradation of Cdk5 or p35.

Among the isolated cDNA clones, we chose SCLIP and CRMP-1 for further analysis, since both proteins are known to function in the central nervous system.

Cdk5 can phosphorylate SCLIP

SCLIP (SCG10-like protein; stathmin 3) is a member of the stathmin family. SCG10 (stathmin 2) is highly concentrated in developing neurons and can inhibit microtubule polymerization and induce depolymerization (Riederer et al. 1997). SCLIP is expressed in mature neurons and can also depolymerize microtubules in vivo (Gavet et al. 1998; Ozon et al. 1998). The function of the stathmin family members is regulated by phosphorylation, and the amino acid sequences encompassing the known phosphorylation sites are highly conserved among them. Different kinases phosphorylate these sites in an overlapping fashion: MAP kinase can phosphorylate Ser62 and Ser73 of SCG10, whereas only one major site (Ser73) is phosphorylated by Cdks including Cdk5 (Antonsson et al. 1998) (Fig. 2A). In the case of stathmin 1, two sites, Ser25 and Ser38, are phosphorylated by Cdks (Marklund et al. 1993) (Fig. 2A). Since SCLIP has the equivalent site to Ser73 of SCG10 with several other potential phosphorylation sites, we investigated whether Cdk5 could phosphorylate SCLIP in two ways, in vitro phosphorylation and a band-shift by phosphorylation in cultured cells. For in vitro phosphorylation, we first purified bacterial SCLIP protein for the substrate (Fig. 2B) and a partially purified human Cdk5/p25 complex as the kinase source. Phosphorylation of recombinant SCLIP was observed only when Cdk5/p25 was present (Fig. 2C, lane 2), and the signal was not detected when roscovitine, a specific inhibitor of Cdk5, was added to the reaction mixture (lane 4), indicating that the recombinant SCLIP was phosphorylated in vitro in a Cdk5-dependent fashion. Next we carried out a similar experiment using a partially purified human Cdk5/p35 complex and the Flag-tagged WT or mutant SCLIP that had been immunoprecipitated from cultured cells as the kinase source and substrate, respectively. As shown in Fig. 2D, phosphorylation of SCLIP was detected in the presence of Cdk5/p35 (Fig. 2D, lane 4), and the signal was not observed in the absence of the kinase complex (lane 3) or the SCLIP substrate (lanes 1 and 2) or with mutant SCLIP in which Ser at 60, 65, 68 and 70 were all replaced by Ala (lane 6), indicating that these Ser residues were important for phosphorylation of SCLIP in vitro. Cdk5 phosphorylated p35 when Cdk5/p35 was present in the reaction mixture (lanes 2, 4, and 6).


Figure 2
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  		Figure 2  SCLIP is a novel substrate for Cdk5. (A) Alignment of the mouse and human stathmin family members including stathmin 1, stathmin 2 (SCG10), and stathmin 3 (SCLIP) with designation of identified phosphorylation sites by solid boxes while putative phosphorylation sites are boxed with a broken line. The numerals above the amino acid sequences indicate the numbers of amino acid residues taking the initiating Met as +1. (B) Purity of bacterial SCLIP protein was tested by an SDS polyacrylamide gel electrophoresis and by staining with Coomassie Brilliant Blue (CBB) or by immunoblotting with anti-thioredoxin antibody (WB). (C) Bacterially purified SCLIP fusion protein was subjected to the phosphorylation reaction using [{gamma}-32P]-ATP and Cdk5/p25, and in the presence or the absence of roscovitine as indicated. Proteins were separated by SDS-PAGE and then stained with CBB. Phosphorylated protein was detected by autoradiography (ARG). (D) The WT and the quadruple mutant (SA) SCLIP were immunoprecipitated and subjected to phosphorylation reaction using [{gamma}-32P]-ATP and Cdk5/p35. The bands of phosphorylated protein corresponding to p35 or SCLIP are designated on the left-hand side of the panel. (E) Immunodepletion of SCLIP from the cell lysate was demonstrated by immunoblotting of the supernatant fraction after the immunoprecipitation reaction of the lysate with or without anti-Flag antibody. (F) The WT and quadruple or triple mutant of SCLIP were subjected to in vitro phosphorylation reaction as described in (D) (ARG). For mutant SCLIP, S60, Ser65/68/73Ala; S65, Ser63/68/73Ala; S68, Ser63/65/73Ala; and S73, Ser63/65/68Ala mutant. The amounts of the input of each protein to the reaction were analyzed by immunoblotting (WB). (G) Western blot analysis of Flag-SCLIP (WT or the quadruple mutant [SA]) expressed alone or co-expressed with Cdk5 or dominant-negative (DN) Cdk5 and p35 in COS-7 cells as designated. Flag-SCLIP, Cdk5, and p35 were probed with anti-Flag, anti-Cdk5, and anti-p35 antibodies, respectively. The upper arrow on the right-hand side of the panel indicates SCLIP that showed a slower mobility in the presence of active Cdk5/p35 (lane 3). (H) Western blot analysis of Flag-SCLIP (the WT, quadruple [SA] or triple SCLIP mutant [S60, S65, S68, or S73]; lanes 1–6), and the effect of roscovitine (Ros) on the band-shift of the WT SCLIP (lanes 7 and 8). The upper arrows on the both sides of the panel indicate the shifted band of SCLIP.

 
We then investigated which Ser residue was responsible for phosphorylation using triple mutants in which one of the four Ser residues in question remained intact. We confirmed successful enrichment of Flag-SCLIP in the immunoprecipitated fraction by testing immunodepletion of the fusion protein from the cell lysate, since Flag-SCLIP migrated on an SDS-polyacrylamide gel very close to the IgG light chain, which made it very difficult to detect the immunoprecipitated Flag-SCLIP by immunoblotting. As shown in Fig. 2E, Flag-SCLIP was not detected in the supernatant fraction obtained after the immunoprecipitation reaction while it was present in the fraction when the immunoprecipitation was done without anti-Flag antibody. In the mock sample where only the Flag vector was introduced, no radioactive signal was detected by in vitro phosphorylation (Fig. 2F, lane 1 of the upper panel), suggesting again that phosphorylation was depending on the presence of SCLIP. The WT SCLIP was phosphorylated whereas the quadruple mutant was not (Fig. 2F upper panel, lanes 2 and 3), and all of the triple mutants were phosphorylated being S73 most weakly (Fig. 2F, lanes 4–7). These proteins were present at an almost similar level to each other (Fig. 2F, lower panel). Since no phosphorylation signal was detected in the mock fraction or in the quadruple mutant, these results indicate that the observed signal was dependent on the Ser residue of SCLIP; that is, the four Ser residues were the targets of Cdk5 in vitro with a different efficiency.

To test in vivo phosphorylation of SCLIP, a mobility shift of Flag-SCLIP co-expressed with Cdk5/p35 in COS-7 cells was analyzed by Western blotting (Fig. 2G). COS-7 cells gave a clearer shift of SCLIP than HEK293 cells for an unknown reason. A significant mobility-shift of SCLIP (indicated by the upper arrow on the right-hand side of the panel) was observed when Cdk5/p35 were overproduced (Fig. 2G, lane 3). Since COS-7 cells have endogenous Pro-directed kinases including other Cdks and MAP kinases, a modest band-shift of SCLIP was observed without overproduction of Cdk5/p35 (lane 2). When dominant-negative (DN) Cdk5 in which Thr replaced Lys at 33 (Nikolic et al. 1996) was overproduced instead of the WT Cdk5, the intensity of the shifted band was decreased (lane 4), indicating that the observed mobility-shift was specifically dependent on the activity of Cdk5/p35. This conclusion was further confirmed by a treatment with roscovitine: when this drug was added to the culture medium, the band-shift of the WT SCLIP was greatly reduced (Fig. 2H, lanes 7 and 8). The observed weak shift in the presence of roscovitine could be due to endogenous Pro-directed kinases as described above. When the quadruple mutant was expressed, no band-shift was observed regardless of the presence of Cdk5, either WT or DN (Fig. 2G, lanes 6–8). With respect to the triple mutants, a modest band-shift was observed in the mutant where Ser68 remained intact, while it was more prominent in the mutant where Ser73 was intact (Fig. 2H, lanes 5 and 6). The other triple mutants and the quadruple mutant did not exhibit a detectable shift (Fig. 2H, lanes 2–4). These results indicate that the shift was highly dependent on Cdk5 activity, and suggest that Ser 68 and Ser73 were the target of Cdk5 in vivo although the latter was a poor substrate in vitro. It is still possible that Ser60 and Ser65 were in vivo target without accompanying a band-shift.

Cdk5 can phosphorylate CRMP-1

CRMP (Collapsin response mediator protein) was initially identified as a mediator of semaphorin-induced growth-cone collapse, possibly linking the semaphorin signaling receptors to small G proteins (Goshima et al. 1995). CRMP-2 is crucial for determining the fate of the axon and dendrites, thereby establishing and maintaining a neuronal polarity, and it binds to tubulin heterodimers to promote the microtubule assembly, regulating the axonal growth and branching (Fukata et al. 2002). Recently, CRMP-2 was reported to be phosphorylated at Ser522 by Cdk5 (Brown et al. 2004), and the phosphorylation of this residue primes for subsequent phosphorylation at Thr514 and Ser518 by GSK3ß that is also a kinase activated in response to semaphorin 3A (Cole et al. 2004; Yoshimura et al. 2005). Since the amino acid sequence encompassing Ser522 is highly conserved between CRMP-1 and -2 (Fig. 3A), it is likely that Cdk5 also phosphorylates CRMP-1. To test this idea, we analyzed phosphorylation of CRMP-1 in vitro and in cultured cells as in the case of SCLIP.


Figure 3
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  		Figure 3  Cdk5 can phosphorylate CRMP-1 at T509 and/or S522. (A) A schematic representation and alignment of the human (h) and mouse (m) CRMP-1 and CRMP-2 C-terminal sequences. The known phosphorylation sites in CRMP-2 by GSK3ß and Cdk5 are shadowed and boxed, respectively, and dotted boxes designate possible phosphorylation sites in CRMP-1. (B) In vitro phosphorylation of the WT (lanes 1 and 2) or double mutant (AA, lanes 3 and 4) {Delta}N433 protein. The GFP-tagged proteins were immunoprecipitated from HEK293 cell lysate with anti-GFP antibody as indicated and subjected to the phosphorylation reaction in the presence or the absence of Cdk5/p25 as indicated. GFP (lanes 5 and 6) and tau protein (lanes 7 and 8) were also used as a substrate for the kinase reaction. The positions of the molecular weight marker proteins are designated on the left-hand side of the panel. (C) In vitro phosphorylation of the WT (lanes 1 and 2) or mutant (lanes 3 and 4) full length CRMP-1. The GFP-tagged proteins were immunoprecipitated and subjected to the phosphorylation reaction as described in (B). AATYK1 was used as a control substrate (lanes 5 and 6). The positions of the molecular weight marker proteins are shown as in (B). (D)In vitro phosphorylation (ARG) and Western blot of the immunoprecipitated fraction (WB) of the WT (lane 2) and double (AA, lane 3) or single (AS for Thr509A and TA for Ser522Ala mutant, lanes 4 and 5) {Delta}N433 mutant protein expressed in COS-7 cells. (E) Western blot analysis of the WT and AA mutant {Delta}N433 proteins (left panel, lanes 1–7) and the full-length of WT or mutant CRMP-1 (right panel, lanes 8–14). GFP-CRMP-1, Cdk5, and p35 were probed with anti-GFP, anti-Cdk5, and anti-p35 monoclonal antibodies, respectively. The arrow in each panel designated {Delta}N433 or full-length CRMP-1 protein that showed a slower mobility. (F) A band-shift of the WT or mutant {Delta}N433 proteins (AA, AS or TA) was analyzed by Western blotting (left panel, lanes 1–4), and the effect of roscovitine (Ros) on the band-shift of the WT CRMP-1 {Delta}N433 (lanes 5 and 6). The upper arrows on the both sides of the panel indicate the shifted band.

 
The CRMP-1 cDNA clone obtained by this screening encoded the C-terminal portion (aa 434–572) of CRMP-1 containing Ser522. Therefore, we cloned cDNA encoding the full-length CRMP-1 by PCR, and used both the full-length and truncated CRMP-1 ({Delta}N433) for analysis. In CRMP-1 as well as CRMP-2, there is Thr509 preceding Pro, which comprises a possible phosphorylation site by Cdk. These Ser and Thr residues of CRMP-2 are among those hyperphosphorylated in Alzheimer's disease where Cdk5/p35 activity is deregulated (Patrick et al. 1999; Gu et al. 2000). We first asked whether Cdk5 phosphorylated CRMP-1 in vitro and whether Ser522 and/or Thr509 of CRMP-1 were susceptible to phosphorylation. For this purpose, a CRMP-1 mutant in which Ala replaced these two residues was constructed for both of the full-length and truncated proteins. We tried to purify CRMP-1 from bacteria by using several different tags, but were unable to produce the recombinant protein to a sufficient level. Therefore, we started in vitro phosphorylation experiment using cultured cells. The GFP-tagged WT or mutant CRMP-1 proteins were immunoprecipitated from HEK293 cell lysate with anti-GFP antibody and subjected to the phosphorylation reaction using partially purified Cdk5/p25 (Fig. 3B,C). Cdk5/p25 could phosphorylate tau (Fig. 3B lane 8) and AATYK1 (apoptosis-associated tyrosine kinase 1; Fig. 3C, lane 6), both of which are known substrates of Cdk5, indicating the Cdk5/p25 complex used in this reaction was active. Both of {Delta}N433 and the full-length CRMP-1 proteins were phosphorylated efficiently by Cdk5/p25 (lane 2 of Fig. 3B,C, respectively), whereas the phosphorylation signal of T509A S522A (AA) mutant of the truncated or the full-length CRMP-1 proteins was not observed to a detectable level (lane 4 of Fig. 3B,C, respectively). Similarly, GFP did not give a detectable signal in the presence of Cdk5/p35 (Fig. 3B, lane 6). Taken together, these results indicate that neither GFP nor protein(s) other than GFP-CRMP-1 in the immunoprecipitated fraction was responsible for the observed phosphorylation signal. To study which residue receives phosphorylation by Cdk5, we constructed single mutants of {Delta}N433 in which either Thr509 or Ser522 was replaced by Ala (AS or TA). As shown in Fig. 3D, a phosphorylation signal was observed in WT (lane 2), AS and TA mutants (lanes 4 and 5) but not in AA mutant (lane 3) or in GFP alone (lane 1), indicating that Cdk5 acted on Thr509 and Ser522 residue of CRMP-1. The lower panel of Fig. 3D representing the immunoblot of the immunoprecipitated samples showed that all of the WT and mutant CRMP-1 proteins tested were successfully immunoprecipitated, confirming that the observed phosphorylation signal was specifically dependent on the presence of the Thr509 and Ser522 residues of CRMP-1.

A mobility shift of CRMP-1 by phosphorylation of the WT and mutant of both of the full-length and {Delta}N433 proteins tagged with GFP was analyzed by Western blotting (Fig. 3E). With respect to the {Delta}N433 protein (Fig. 3E, left panel), a band-shift (indicated by the arrow) was observed in the presence of Cdk5/p35 (Fig. 3E, lane 3), but DN Cdk5 failed to generate the shift (lane 4). Cdk5/p35 failed to induce a mobility-shift of the {Delta}N433 AA mutant (lane 6), suggesting that Cdk5/p35 and the Ser or Thr residue were responsible for the observed shift. Similar results were obtained with the wild-type full-length CRMP-1 protein (Fig. 3E, right panel). When roscovitine was present in the culture medium, a band-shift of the WT{Gamma}{Delta}N433 protein was diminished (Fig. 3F, lanes 5 and 6), confirming that the shift was dependent on the Cdk5 activity. Next, we investigated which residue, Thr509 or Ser522, was responsible for the observed band-shift. As shown in Fig. 3F, the shift was observed with the WT and TA mutant (Fig. 3F, lanes 1 and 4), but not in AA or AS mutant (lanes 2 and 3), suggesting that phosphorylation at Thr509 played a major role in the band-shift.

Mutant SCLIP or CRMP-1 failed to suppress growth defect caused by overproduction of Cdk5/p35

Finally, we tested the ability of the mutant SCLIP and CRMP-1 proteins to suppress the growth defect of the MFY188 strain overproducing Cdk5/p35. If our assumption that overproduction of a substrate of Cdk5 can suppress the growth defect is correct, the mutant proteins that were not phosphorylated by Cdk5 should fail to restore the growth of the yeast cells overproducing Cdk5/p35. The results shown in Fig. 4 indicates that that was exactly the case: MFY188 overproducing the WT CRMP-1 or WT SCLIP, together with Cdk5/p35 could grow on a galactose plate, whereas neither those producing the mutant proteins in which all Ser or Thr residues were replaced by Ala nor MFY188 harboring vector alone could grow (Fig. 4, upper panel). We also tested an overproduction effect of another set of mutant CRMP-1 or SCLIP proteins in which all Ser or Thr residues in question were changed to Glu or Asp to mimic phosphorylation of these sites. As shown in Fig. 4, neither of these mutants could suppress the growth defect of their host cells overproducing Cdk5 and p35, suggesting that the presence of negative charges at these sites was not able to recover the growth of the yeast cells. Thus it is highly likely that phosphorylation of the Ser and Thr residues in CRMP-1 and SCLIP by Cdk5 is required for suppression of the growth defect.


Figure 4
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  		Figure 4  Suppression of growth defect caused by overproduction of Cdk5 and p35 in yeast cells. MFY188 cells transformed with vector, GAL-CRMP-1 (WT), GAL-CRMP-1 T509A S522A (Mut AA), GAL-SCLIP (WT), GAL-SCLIP S60A S65A S68A S73A (Mut SA), GAL-CRMP-1 T509E S522E (Mut EE), or GAL-SCLIP S60D S65D S68D S73D (Mut SD) were streaked on the SGalRaf medium, and the growth was scored after incubating the plate at 36.5 °C for 4 days.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
By using a yeast system, we could discover novel substrates for Cdk5, SCLIP and CRMP-1. This conclusion was derived from three lines of evidence: in vitro phosphorylation, a mobility-shift by in vivo phosphorylation and the requirement of phosphorylatable residue(s) for suppression of the growth defect of yeast cells. We also revealed that specific Ser and Thr residues of SCLIP and CRMP-1 were the target of Cdk5 in vitro and possibly in vivo. SCLIP and SCG10 are members of the stathmin family, and both proteins can depolymerize microtubules in vivo, and the action of SCG10 on microtubules is controlled by phosphorylation of Ser73 by kinases including Cdk5 (Antonsson et al. 1998). We demonstrated that Cdk5 phosphorylated Ser73 of SCLIP both in vivo and in vitro, making it highly likely that Cdk5 regulates the interaction of SCLIP with microtubules by phosphorylation. The CRMP family is critical to Sema3A signaling and Cdk5 phosphorylates Ser522 of CRMP-2 (Brown et al. 2004). Functional analysis with a mutant CRMP-1 revealed that CRMP-1 is also functioning in PlexinA1-dependent Sema3A signaling (Deo et al. 2004). A recent paper reported that the CRMP family is involved in the neurite outgrowth controlled by neurotrophins: CRMP-1 is involved in the neurite extension induced by neurotrophin 3 (NT3) whereas CRMP-2 is required to suppress that induced by NGF (Quach et al. 2004) and the Cdk5 activity appears to be stimulated after the treatment of neurons with NT3 (Tokuoka et al. 2000). While we were revising this manuscript, Cole et al. (2006) reported that Cdk5 could phosphorylate mouse CRMP-1 at Thr509. Taken together, these results are consistent with our conclusion that Cdk5 can phosphorylate CRMP-1 at Thr509 and Ser522 in vitro, and at least Thr509 in vivo.

As shown in Table 1, there are more candidates for substrates of Cdk5, including cDNAs with unknown function. Since this yeast screening system is not time-consuming, it enables extensive as well as efficient isolation and identification of novel substrates for Cdk5. Moreover, using a cDNA expression library prepared from mouse whole body will enable us to identify Cdk5 substrates that are phosphorylated in tissues other than the nervous systems. Among the cDNAs listed in Table 1, those with known function did not appear to encode regulators of Cdk5/p35, but those with unknown function could encode such regulators. It is possible that some cDNAs isolated by this screening could suppress or activate a downstream event(s) that was compromised by hyperphosphorylation of yeast proteins by Cdk5/p35. We tested whether Skp1 or a cDNA product (accession number CO804842) could be phosphorylated by Cdk5 or could inhibit Cdk5 activity on SCLIP in vitro, but failed to detect phosphorylation of these proteins or a significant reduction in Cdk5 activity (data not shown). We could imagine that these proteins could recover the yeast growth by modulating the downstream events.

This screening system can also be applied to identification of activators of Cdk5/p35: using a yeast strain that over-expresses Cdk5 but produces p35 at a low level, introduction of a mouse cDNA library and subsequent screening of yeast clones that fail to grow on the selective medium will provide cDNA clones which can stimulate Cdk5/p35 activity. Furthermore, when appropriately modified to incorporate small peptide or chemical compound, the yeast cells overproducing Cdk5/p35 can be used to screen a peptide or drug that inhibits Cdk5/p35 activity. Further screening with these systems will enable us to identify more novel substrates and regulators of Cdk5/p35, which will lead to exploration of Cdk5 function in diverse cellular systems and especially in the mechanisms of neurodegenerative diseases.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Strains, media and cell culture

MFY188 (MAT{alpha} GAL+ trp1 his3{Delta}200 pep4::HIS3 prb1{Delta} leu2::GAL-Cdk5-TRP1 ura3::GAL-p35-LEU2) was derived from BJ5464 strain by integrating fragments expressing Cdk5 or p35 under the control of the GAL promoter. Briefly, a leu2 fragment that has an insertion containing the GAL promoter and mouse Cdk5 cDNA derived from pMF1086 plasmid (Nishizawa et al. 1999a) and a TRP1 marker between ClaI and SalI sites of a fragment, and a URA3 fragment that has an insertion of a fragment containing the GAL10 promoter and mouse p35 cDNA and a LEU2 marker between NcoI and EcoRV sites were constructed and were integrated into leu2 and ura3 loci of BJ5464 strain, respectively. Successful integration and production of recombinant proteins were confirmed by PCR (data not shown) and Western blotting (Fig. 1B, lane 1), respectively. Yeast cells were grown at 30 °C in SD medium containing 0.67% Difco Yeast Nitrogen Base, 2% Glucose, and appropriate nutritional supplements or in SGalRaf medium where 2% each of galactose and raffinose replace glucose in SD medium (Rose et al. 1990). Escherichia coli DH5{alpha}+ was used as a host for plasmids. HEK293 or COS-7 cells used for analysis of mouse brain proteins were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich, Poole, UK) supplemented with 10% fetal bovine serum, 100 units/mL penicillin and 0.1 mg/mL streptomycin at 37 °C in a 5% CO2 incubator.

Screening of mouse cDNA library

Standard E. coli and yeast genetic methods were as previously described (Rose et al. 1990; Sambrook & Russell 2001). Mouse brain cDNA expression plasmid library was constructed by introducing mouse brain cDNA (TaKaRa, Kyoto, Japan) between ClaI and BglII sites of pMF1129 plasmid that had the GAL10 promoter and the TDH3 terminator in a YEp-URA3 backbone to express mouse cDNA under the control of the GAL10 promoter. MFY188 cells were transformed with a mouse brain cDNA library in pMF1129 plasmid by high-efficiency yeast transformation method (Gietz & Woods 2002), and resuspended in SD medium lacking leucine, tryptophan and uracil for 5–6 h at 30 °C to establish transformants. Production of Cdk5, p35, and mouse brain protein were then induced by transferring the cells to SGalRaf medium supplemented with appropriate nutrients and incubation at 30 °C for 16 h. Then the cells were plated onto a SGalRaf plate, and incubated at 30 °C for 16 h, followed by incubation at 36.5 °C for 5–7 days. The higher temperature was effective to reduce background level, i.e. to pronounce the deleterious effect of overproduction of Cdk5 and p35. The colonies that formed on the selection medium were picked up, transferred to the same fresh plate, and incubated at 36.5 °C again to confirm the recovery of growth.

The cDNA plasmids in the transformants that could grow on the selection medium were recovered into E. coli, and those plasmids having a sufficient length of cDNA (more than c. 500 bp) were selected to transform into MFY188 again to confirm their ability to recover growth of yeast. Those confirmed cDNA clones were then subjected to DNA sequencing using primers 5'-GGTGGTAATGCCATGTAATATGAT-3' and 5'-GTCGACTCAATCAATCACAGCTAGC-3' as forward and reverse primers, respectively, and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Tokyo, Japan). Obtained sequences that had a sufficient length of ORF were referred to the GENBANK database whether homologous sequence was present.

Construction of expression plasmids

For expression in cultured cells, cDNA encoding the entire CRMP-1 ORF was cloned by PCR from the mouse brain cDNA library using primers MN428 (5'-CCACAGGGGCCATGGCTCATCAGGGGAAG-3') and MN463 (5'-CTCTTGCATATCTGGCTCGTCGACGTCAACCGAGGCTGGTG-3') to incorporate NcoI and SalI as cloning sites, respectively. To remove a poly(A) tail from the initially cloned CRMP-1 C-terminal fragment that lacks first 433 amino acids, {Delta}N433 CRMP-1 cDNA was cloned by PCR using pSP72 containing the CRMP-1 C-terminal fragment as template and primers MN442 (5'-GGAAACATCAGTGTCAGCAAGATCTTGGGCCGCTTCATCCCTCGG-3') and MN463 to incorporate BglII and SalI as cloning sites, respectively. Similarly, cDNA encoding SCLIP ORF was cloned by PCR using primers MN754 (5'-GCCGCCGCCAGCACCATGGCCAGCACCG-3') and MN755 (5'-GTTCTTGTCGCCGCTGGGTCGAATTCTCCTTAGCCAGACATTTC-3') to incorporate NcoI and EcoRI sites, respectively. The sequences of the cloned cDNAs were confirmed by DNA sequencing. The PCR products were purified with a QIAquick PCR purification Kit (Qiagen) and cleaved with appropriate restriction enzymes, and CRMP-1 and SCLIP cDNAs were then incorporated into pEGFP (Clontech, Oxford, UK) and pFLAG-CMV2 (Sigma, St. Louis, MO, USA) plasmids, respectively.

To mutate putative phosphorylation sites in SCLIP at serine 60, 65, 68 and 73 to alanine or to aspartic acid, and in CRMP-1 at threonine 509 and serine 522 to alanine or to glutamic acid, site-directed mutagenesis was carried out using a Quick Change site directed mutagenesis kit (Stratagene Europe, Amsterdam, the Netherlands), and the following mutagenic primer pairs (the sequence of each primer set is complementary): 5'-GAGCTTCGAGGTCATCCTCAAGGCTCCTTCTGACCTAGCTCCAGAGGCCCCTGTGCTCTCTGCTCCTCCCAAGAGGAAGGATGC-3' for SCLIP SA (all Ser to Ala), 5'-GAGGTCATCCTCAAGGATCCTTCTGACCTAGATCCAGAGGACCCTGTGCTCTCTGATCCTCCCAAGAGG-3' for SCLIP SD (all Ser to Asp), 5'-GTGCCAGCTGCACCCAAACAT-3' for CRMP-1 AS (Thr509Ala), 5'-GCCAAATCCGCGCCTTCTAAA-3' for CRMP-1 TA (Ser522Ala), 5'-GTACGAGGTGCCAGCTGAACCCAAACATGCTGCTCCTGCTCCTTCTGCCAAATCCGAGCCTTCTAAACACCAAC-3' for CRMP-1 EE (Thr/Ser to Glu) according to the manufacturer's protocol. To produce SCLIP mutants in which three of four Ser residues in question were replaced by Ala, the SCLIP SA mutant was used as template with following primer pairs having complementary sequences: 5'-GAGGTCATCCTCAAGTCTCCTTCTGACCTA-3' for S60 (Ser65/68/73Ala), 5'-CTCCTTCTGACCTATCTCCAGAGAGCCCT-3' for S65 (Ser60/68/73Ala), 5'-GACCTATCTCCAGAGAGCCCTGTGCTCTC-3' for S68 (Ser60/65/73Ala), and 5'-GCCCTGTGCTCTCTTCTCCTCCCAAGAGG-3' for S73 (Ser60/65/68Ala). The successful conversion was verified by DNA sequencing. The WT or mutant CRMP-1 and SCLIP cDNAs were also incorporated into pMF906 plasmid (Nishizawa et al. 1999b) to be expressed under the GAL10 promoter, and the expression units were then transferred to pRS316 plasmid to construct pMF1442 (WT CRMP-1), pMF1443 (WT SCLIP), pMF1451 (CRMP-1 AA mutant), pMF1452 (SCLIP SA), pMF1481 (CRMP-1 EE), and pMF1482 (SCLIP SD).

Purification of bacterial SCLIP

SCLIP cDNA was incorporated into pThioHis B plasmid (Invitrogen) between NcoI and EcoRI sites, and induction of His-thioredoxin (His-Thio)-SCLIP fusion protein in E. coli Top10 cells and preparation of bacterial cell lysate were carried out as described (Nishizawa et al. 1999b). The fusion protein was absorbed to an affinity column of the ProBond resin (Invitrogen), and was eluted with 20 mM sodium phosphate buffer, pH 6.0, containing 0.1% Triton X-100, 500 mM NaCl, 60 mM imidazole.

Purification of human recombinant Cdk5/p25 or p35 complex

Human Cdk5 cDNA or His6-tagged p35 or p25 cDNA (a deletion product of p35), in a baculovirus transfer vector, BacPAK9 (Clontech, Palo Alto, CA, USA), was constructed as previously described (Hashiguchi et al. 2002), and these plasmids were co-transfected with BacPAK6 virus DNA (Clontech) into Sf9 cells following the manufacturer's instructions. Recombinant Cdk5/p35 or p25 complex was purified essentially as described (Sakaue et al. 2005). Briefly, frozen Sf9 cells were resuspended in cold extraction buffer (10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 1% Triton X-100, 10 mM imidazole, 10 mM ß-mercaptoethanol, 0.2 mM Pefabloc SC, 1 µM E-64, 10 µg/mL leupeptin), and sonicated 3 times for 10 s at 4 °C, followed by centrifugation at 19 000 g for 15 min at 4 °C to obtain cleared lysate. The Cdk5/p35 or p25 complex in the lysate was bound to a nickel-nitrilotriacetic acid-agarose column, and was eluted with 10 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl and 350 mM imidazole.

Immunoblot analysis

Immunoblot analysis was carried out essentially as previously described (Nishizawa et al. 1999b). Production of Cdk5 and p35 proteins in yeast cells was induced by incubation in SGalRaf medium supplemented with appropriate nutrients at 30 °C for 30 h. The cells were then suspended in 20 mM phosphate buffer, pH 7.2, containing 150 mM NaCl, 10 mM KCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail (2.5 µg/mL each of aprotinin, leupeptin, pepstatin A and anti-pain 50 µg/mL each of L-1-tosylamide-2-phenylethylchloromethyl ketone and Na-p-tosyl-L-lysine chloromethyl ketone), and were disrupted by vortexing with glass beads, and the extracts were cleared by centrifugation at 12 000 g for 5 min at 4 °C. Transfection of Cdk5 or p35 in pCMV plasmid, SCLIP in pFlag-CMV2 plasmid, or CRMP-1 in pEGFP plasmid into HEK293 or COS-7 cells was performed using Polyfect transfection reagent (Qiagen) according to the manufacturer's protocol. After 24 h, cells were washed twice with PBS, collected by centrifugation, and subjected to sonication to obtain cell lysates.

Proteins were separated on an SDS polyacrylamide gel and electrotransferred on to a nitrocellulose membrane, and the blot was probed with anti-Cdk5 monoclonal antibody at a 1 : 250 dilution (Patrick et al. 1999), anti-p35 polyclonal antibody at a 1 : 1000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag M2 monoclonal antibody at a 1: 2000 dilution (Sigma), or anti-GFP monoclonal antibody at a 1 : 1000 dilution (Roche Diagnostics, Mannheim, Germany). After the membrane was rinsed with TTBS 3 times for 10 min each, the proteins were probed by incubating with horseradish peroxidase-conjugated goat antibodies to rabbit or mouse immunoglobulin G (ICN Pharmaceuticals, Aurola, OH, USA). The proteins were then visualized with a Renaissance chemiluminescence system (New England Nuclear, Boston, MA, USA) and the immunoblot images were captured using a LAS3000 image analyzer (Fujifilm, Tokyo, Japan).

In vitro kinase assay

Cleared HEK 293 containing either the wild-type or mutant GFP-CRMP1 fusion protein in 25 mM HEPES, pH 7.5, containing 1% Triton X-100, 0.3 M NaCl, 1 mM MgCl2, 1 mM EGTA, 5% glycerol (HEPES buffer) were incubated with anti-GFP antibody on ice for 1 h. Protein G-Sepharose 4 Fast Flow (Amersham Bioscience) was added to the mixture, which was then incubated at 4 °C for 1 h, followed by washing 3 times with HEPES buffer, and twice with kinase assay buffer (20 mM Tris.HCl, pH 7.5, 7.5 mM MgCl2, 1 mM ZnSO4). Similarly, cleared HEK293 cell lysates containing either the wild-type or mutant Flag-SCLIP fusion protein in 50 mM Tris.HCl, pH 7.5, 150 mM NaCl, 1 mM sodium pyrophosphate, 0.1% SDS, 1% Triton X-100, 1% deoxycholate (RIPA buffer) were incubated with anti-Flag antibody M2 (Sigma Aldritch), followed by immunoprecipitation with Protein G-Sepharose 4 Fast Flow, and by washing as described above. The beads were then resuspended in 40 µL of the kinase assay buffer containing 0.1 mM ATP, 5 µCi of [{gamma}-32P]ATP, and 0.5 µg/mL recombinant Cdk5/p35. The reaction mixture was incubated at 30 °C for 30 min, and the reaction was stopped by adding 4 x SDS loading buffer and the proteins were subjected to electrophoresis on an SDS polyacrylamide gel and were analyzed by autoradiography with a BAS5000 Bioimage Analyzer (Fujifilm).


   Acknowledgements
 
We are grateful to G. Patrick and L.-H. Tsai for anti-Cdk5 monoclonal antibody. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (S. H., A. T., and M. N.) and by Keio Gijuku Academic Development Funds (M. N.).


   Footnotes
 
Communicated by: Masayuki Yamamoto

* Correspondence: E-mail: mas{at}sc.itc.keio.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 13 January 2006
Accepted: 20 September 2006




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