Karcher and colleagues (2001)121 showed that incubation of melanosomes with
mitotic cell extract from Xenopus l. oocytes leads to the release of melanosome- associated Myosin V. In addition, the authors provided evidence that the observed release was due to Calcium-/Calmodulin-dependent Kinase II-α
(CaMKII-α)-mediated phosphorylation of a specific Serine residue within the tail
domain of Myosin V. With this in mind, I decided to take a closer look at the role of CaMKII-α in melanosome transport.
Therefore, my primary goal was to set up an assay where melanosomes that contain Myosin Va are targeted for CaMKII-α-mediated release in vitro. Such a
method would for the first time allow for the specific manipulation of melanosome transport in vitro. Thus it would be finally possible to determine in vitro using a native cellular cargo, if and how Myosin V might assist the Kinesin-2-driven cargo transport on microtubules (for a detailed protocol, refer to Section 4.3.5.6).
5.2.1 Results
5.2.1.1 Cloning and purification of baculovirus-expressed CaMKII-α
Most studies that in the past dealt with the characterization of CaMKII, used protein that had been purified from rat brain, simply because in the forebrain CaMKII composes up to 1% of the total protein 168. However because of twofundamentally important reasons, in this work I aimed on setting up a protocol for the purification of baculovirally expressed recombinant CaMKII-α: i) enhancing
the yield of total kinase protein, while lowering the required labor time; and ii) not being relient on supply of rat brain as a natural source. This section provides a brief summary of the cloning, and purification procedure of baculovirus-expressed CaMKII-α.
The coding sequence of CaMKII-α (kindly provided by Prof. Y. Hayashi, RIKEN
institute, Saitama, Japan), was originally blunt-end cloned into the pBluescript SK (-) vector via EcoRI. Therefore, the coding sequence, which at this point still also contained a portion of non-coding region (approx. 200 bases), was retrieved via
To amplify the coding region from the template sequence and to add the sequence encoding for the FLAG-tag (either to the 3’ or 5’ ends), PCR was carried out (for a complete list of primers as well as details on the specific PCR performed, see Sections 3.4 and 4.2.2.2.1). An N’, C’FLAG-tagged as well as an untagged version containing SpeI- (3’) and NotI-restriction sites (5’), were generated (Figure 42 B). The two FLAG-tagged constructs are 1470 bp long, the
Figure 42. Cloning of FLAG-tagged CaMKII-α for baculovirus expression.
(A) The CDS (and approx. 200 bases non-coding region) encoding for CaMKII-α was retrieved
from the pBluescript SK (-) vector by restriction enzyme-mediated digest with EcoRI. The purified fragment of size 1673 bp subsequently was sequenced and used as template during further cloning. (B) The sequence encoding for the FLAG-tag motif was fused 5’ and 3’ of the CaMKII-α gene, by using forward and reverse primers containing respectively SpeI- and NotI-
restriction sites along with the FLAG-encoding sequence. The amplified product (approx. size 1500 bp) was used for the subsequent cloning into the pFastBac 1 vector. (C) Successful cloning of the FLAG-tagged as well as untagged CaMKII-α gene into pFastBac 1 was
confirmed via restriction enzyme-mediated test digestion with SpeI and NotI (left part). A schematic of the insert and vector along with the respective product sizes is provided on the right.
NotI and SpeI, each of the three insert sequences was cloned under the control of the polyhedrin promoter of the baculovirus-specific transfer vector pFastBac 1
(Figure 42 C, see Section 7 for vector maps).
FLAG-affinity purification of the CaMKII-α protein was carried out from 300 to 350
ml of Sf9 cell suspension culture (for details of the purification protocol, refer to Section 4.3.4). Typically concentrations of approx. 20 µM CaMKII-α (corresponds
to approx. 1.1 mg/ml protein) in a final volume of 300 to 400 µl were yielded (Figure 43).
5.2.1.2 Autophosphorylation of CaMKII-α
For CaMKII-α to become an active enzyme, it requires auto-phosphorylation of
Thr286 (T286)169,170. Therefore, it was important to pinpoint under which conditions CaMKII-α autophosphorylation was strong or weak when compared to
the inactive state. For this study, via Western-blot analysis against phosphorylated Thr286 (pT286) of CaMKII-α the kinase’s ability to undergo
autophosphorylation was tested (a detailed protocol can be found in Section 4.3.5.5).
To this end, the kinase was incubated with 300 µM free Ca2+ and ATP (0.1 mM) for different time periods, and auto-phosphorylation was assayed via Western-blot analysis using an antibody against the phosphorylated T286 (pT286) of CaMKII-α
!"#$%$&'($)*+)% !"#$%%&' ,- !"#$%&'( ./01234 5/01234 67 89 77 !"#$%&'()%*%'&$+,$-+!.//01 2)#**#%&'
Figure 43. FLAG-affinity protein purification of N’ and C’FLAG-
tagged CaMKII-α.
Protein was expressed in Sf9-insect cells via the baculovirus expression system, and was FLAG-affinity purified from suspension culture. Cropped images of Coomassie blue-stained SDS polyacryl amide (10%) gels show the eluted fraction of purified N’ and C’FLAG-tagged
CaMKII-α. Both products were
confirmed by mass-spectrometry analysis. Approximate product size is indicated on the right.
As expected, at 300 µM free Ca2+ and 0.01 mM ATP CaMKII-
α showed high
levels of pT286. However, while for the C’FLAG-tagged version the size of the auto-phosphorylated product corresponded to the molecular weight of monomeric CaMKII-α (approx. 55 kDa), for the N’FLAG-tagged construct strong pT286 auto-
phoshorylation was detected in a product of approximately 40 kDa. It should be noted that at the longest reaction time (i.e., three minutes) also at 55 kDa a faint duplex band was detected. In addition, with increasing reaction time (i.e., one to three minutes) the degree of auto-phosphorylation was increased (Figure 44).
Perhaps due to a calcium-SDS gel artifact, the detection levels of total CaMKII-α
protein were higher when Ca2+ was included in the reaction. Judging from the Coomasie-stain (Figure 44), it is unlikely that unequal sample loading accounted
for the higher intensities in the blots. Under zero-Ca2+ conditions, no activation !"#$% !"#$%%&' &!'() !"#$%%&' *""+$,,-. /#$-0 11 23#"&4",&4"56%$#-"0 "7 !"#$%%&' !"#$%&#"'( )*)+,%-+./%�( 12$$%-+./%�( 8 9 : ' 9 8 8;< 8;9 11 =8 11 )) 03%&45+( 8 9 : ' 9 8 8;< 8;9 >?@AB2C *?@AB2C
Figure 44. Autophosphorylation of N’ and C’FLAG-tagged CaMKII-α.
Both, N’FLAG- (left panel) and C’FLAG-tagged (right panel) CaMKII-α, were tested for their
ability to undergo autophosphorylation at T286. For this 270 nM purified kinase protein was stimulated for one to three minutes with 0.1 mM ATP and 0.3 mM free Ca2+ (conditions labeled at the top). Top and middle part: Semi-quantitative Western-blot analysis against total kinase
protein and phosphorylated T286 (pT286). Bottom part: Coomassie blue-stained SDS
polyacryl amide gels (10%) sections showing the kinase bands at 55 kDa. The 66 kDa-product comprises BSA, which was included in the assay. Control (left-most lane): Here, no Ca2+ was included, thus stimulation of the kinase cannot occur. Product sizes (in kDa) are indicated on the right.