Cloning and Sequencing a gene
Cloning and sequencing of cDNA encoding substrates for Ca++ dependent protein kinases.
Benedict A. Gomes, Wei W. Deng and Dr Diana C. Bartelt.
Dec 16 th 1999
(St Johns University-NY-11439)
Abstract:
Phosphorylation screening allows us to isolate only those substrates that are phosphorylated and hence the proteins that match our cloned and sequenced cDNA are probably substrates for protein kinases. We used A. nidulans CaM dependent multifunctional protein kinases (ACMPK) as our phosphorylation model as it shares many functional characteristics as CaMPKII, this kinase has been purified and characterized (Bartelt et al 1988). In the present paper it was shown that the proteins that matched the sequences we isolated, were probable substrates for Ca++ dependent protein kinases. Restriction mapping indicated the presence of the restriction sites in the cDNA.
- Introduction:
Ca ++, as one of the most versatile second messengers involved in basic cellular processes. The intracellular Ca ++ binding protein is mainly calmodulin (CaM) which binds to and activates a great variety of enzymes, such as nucleotide cyclases and phosphodiesterases, nitric oxide synthases and plasma membrane calcium pumps, and most importantly a class of different protein kinases and phosphatase(s), namely, CaM-kinases and calcineurin [4 and 5]. Since protein kinases and phosphatase are important in cellular regulatory processes. CaM-kinases are involved in the regulation of such central processes as neurotransmitter release, muscle contraction, and cell proliferation and gene expression. Six different CaM-dependent kinases are known to date, all belong to the class of enzymes phosphorylating serine threonine residues. The targets can be either of many substrates (e.g., CaM-kinase I, II and IV) or single substrates (e.g., myosin light chain kinase, phosphorylase kinase or CaM-kinase III). Calmodulin ( CAM I, II and IV) dependent multifunctional protein kinases I, II and IV are a set of enzymes that can phosphorylate a wide variety of enzymes and structural proteins, depending on the intracellular Ca ++ content. Ca ++/calmodulin-dependent protein kinase II (CaMKII) is a Ca ++-responsive multifunctional protein kinase, it is known to occur abundantly in the brain [1] and is thought to play important roles in a variety of neuronal functions mediated by Ca ++ [2 and 3]. CaMKII is markedly activated upon autophosphorylation at Thr28. We used A. nidulans CaM dependent multifunctional protein kinases (ACMPK) as our model as it shares many functional characteristics as CaMPKII, this kinase has been purified and characterized (Bartelt et al 1988).
2) Methods and Materials:
2.1 Materials:
l ZAP phage was obtained from stratagene-usa. [ g 32 P] ATP (7000 Ci/mmol) was obtained from Amersham and diluted with unlabelled ATP to get a concentration of 1mCi/ml. ACMPK was used at a concentration of 4µg/ml (Bartelt et el) to carry out the phosphorylation reaction. Rapid excision kit obtained from stratagene was used for plasmid rescue.
2.2 Serial dilution of the library:
Serial dilutions of A. nidulans cDNA library in l was carried out in SM buffer (tris 50mM, NaCl 100mM, gelatin 0.02%, MgSO 4 10mM) and E. coli BB4 colonies, this was pour plated on LB plates till we had a 1000pfu/ml concentration of infected phage.
2.3 Phosphorylation of the plaques:
Using IPTG treated filter papers placed in a blocking solution containing a high concentration of (3%) BSA, two screenings of the library were done at 20,000pfu/ml and 1000pfu/ml. The filters were placed in a wash buffer (20mM tris, 150mM NaCl, 10mM EGTA, 1mM EDTA, 1mM DTT, 0.2mM PMSF, leupeptin 1µg/ml, Pepstatin 0.1µg/ml) containing protease inhibitors and chelating agents for 60mins. They were then placed in phosphorylation buffer (20mM HEPES.KOH, 3mM MgCl 2, 2mM MnCl 2, 50µM Na 3O 4, 2mM DTT, 0.1% TritonX-100) for 10 mins, then phosphorylation buffer+25µM ATP for 1 hr, and then phosphorylation buffer for 10 mins. To these treated filters CaCl 2 (1mM), CaM (6µg/ml), and g 32 P-ATP/ATP mixture (1mCi/ml) were added, then 4µg/ml ACMPK was added for 1hr; the reaction was terminated by washing with wash buffer without the protease inhibitors.
2.4 PCR and plasmid rescue:
Plaques from the second round of screening were cored; put in SM buffer and 5µl of each were mixed with master mix (10x Taq polymerase buffer, dNTP 10mM, T3 promoter 100µM, T7 promoter 100µM, and TAQ DNA polymerase). This was run in a PCR program containing 30 cycles of 40’@ 94 oC, 1’@50 oC, 2’@72 oC, 1 cycle of 10’@72 oC. The amplification products were verified by (1% agarose) gel electrophoresis. Plasmid rescue was done using E coli strains XPORT and XLOLR and 704 helper phage (10 8pfu/ml) using the method described by stratagene (rapid excision kit). The DNA was minipreped on a QIAprep miniprep kit.
2.5 DNA sequencing and restriction mapping:
The plasmid DNA was denatured and precipitated and T7 sequenase and T3 primer were added to generate single stranded DNA. DNA sequencing was then done on a gel (5.7% acrylamide, 0.3% bis acrylamide, 8M urea, 0.089M Tris borate, 0.089M Boric acid, 0.002%M EDTA, 0.01% TEMED, 0.04% Ammonium Persulfate). Restriction mapping of our plasmid DNA sample was done with Eco RI , BamH1, PstI, Sal1, and XbaI restriction enzymes for 1hr at 37 oC and then a gel electrophoresis was done.
3) Results:
3.1 Phosphorylation screening of a phage expression library:
A phage expression vector was constructed using l ZAP cloning vector (fig 1), which has pBluescript phagemid. Our cDNA was inserted in the multicloning site (EcoRI cloning site in our case) present in the LacZ gene this results in the disruption of the LacZ gene, thus the recombinant phages appear white when plated on lac hosts in the presence of IPTG and X-gal while non recombinants are blue in color. Libraries constructed with l ZAP can then be screened with antibody or nucleic acid probes, RNA transcripts can be generated from the T3 and T7 promoters, which flank the cDNA insertion sites. Fragments cloned into l ZAP can be automatically subcloned by excising the cDNA in a single stranded phage, which can then infect an E coli host, where it is propagated as a pBluescript plasmid.
We first screened the plaques for Calmodulin dependent multifunctional protein kinase substrates using ACMPK (4µg) in the presence of CaM (6µg/ml) and g 32 P-ATP/ATP mixture. The plaques were transferred to nitrocellulose filters, which were blocked by treatment with bovine serum albumin (BSA) and then incubated with purified ACMPK in the presence of CaM (6µg/ml) and g 32 P-ATP/ATP mixture. As shown in figure 2 the l phage containing the insert gave strong phosphorylation, whereas the background derived from phosphorylation of l phage or E coli proteins was low thus indicating the presence of substrates for Calmodulin dependent multifunctional protein kinases. We screened the library by phosphorylation at 20,000pfu/ml (Fig 2) for substrates. Serial dilutions were then done till we got 1000pfu’s/ml (refer to methods), and the substrates were confirmed by phosphorylation at 1000pfu/ml (fig 3). All the positive substrates (A to D in fig 3) were then cored and amplified by PCR.
3.2 PCR amplification of selected phage:
All the positive substrates selected from the second round of screening (A to D in fig 3) were amplified by PCR and a gel electrophoresis was done to confirm the presence of substrates. The sample with the largest band and biggest size was selected for plasmid rescue namely lane 9 in fig 4 (sample H1) and lane 10 in fig 5 (sample H1), this corresponds to plaque An in figure 3. From the gel it can be seen that plaques B, C, and D contained DNA of different lengths, hence since we were looking for a pure sample we selected H1 which showed the least presence of other DNA. In fig 4 sample G1-G4 is of a colleague and is not relevant here, in fig 5 samples in lanes 3 to lane 9 are of colleagues. The selected PCR product (sample H1) was then rescued for plasmid DNA.
3.3 Plasmid rescue:
Plasmid rescue was done using E coli strains XPORT and XLOLR and 704 helper phage (10 8pfu/ml) using the method described by stratagene (rapid excision kit). The DNA was minipreped on a QIAprep miniprep kit. This plasmid DNA was then verified by gel electrophoresis, sample H1 3 (lane 3 in fig 6) showed a single clear band and was chosen to be sequenced.
3.4 DNA sequencing/ restriction mapping:
The DNA selected (H1 3) did not show any amino acids. Since the library was being screened by our colleagues too, we shared the information gathered by the other groups F (personal communication from T. Dao & R. Presti) and A (personal communication from J. Hun & S. Rice) figure 7A and 7B gives the cDNA and protein sequences that they screened respectively. Figure 7A corresponds to a transaldolase from the yeast Kluyveromyces lactis. I (figure 7C), it also matches with other transaldolases from different species (yeast, human, mouse etc) and fig 7B corresponds to loricrin from mouse (figure 7D). Sample A3 of group A was also run by us to find the restriction sites (mapping) (fig 8). Lane 7 in fig 8 corresponds to pBluescript, lane 8 corresponds to Pst and shows a band indicating the presence of a pst restriction site, lane 9 shows the presence of an E corI restriction site (where our cDNA was inserted), lanes 10, 11 and 12 show the presence of a XbaI site, Sal1 and BamH1 sites respectively.
4) Discussion:
We used phosphorylation screening to identify physiological substrates for protein kinases, this was done using a purified protein kinase (ACMPK) and a phage cDNA expression library which contained the library of interest to us, this method allowed us to subclone the cDNA as plasmids. Phosphorylation screening has several advantages over other methods for substrate detection; the method of using a library is a powerful tool to identify substrates for a protein kinase (6); and possible substrates can be found by searching gene data banks. However this method though good does not identify substrate proteins directly and proteins containing minimal consensus sequences for a protein kinase may not always be good substrates because of tertiary structure constrains (7).
Apergillus Nidulans comes from the following classification--- Eukaryota; eukaryote crown group; Fungi/Metazoa group; Fungi; Ascomycota; Eurotiales; Trichocomaceae; Emericella (9). The clones that we have isolated correspond to (fig 7A) a transaldolase from the yeast Kluyveromyces lactis. I (figure 7C), and fig 7B corresponds to loricrin from mouse (figure 7D). These two proteins come from the same genus Eukaryota. Transaldolase is an enzyme important in the balance of metabolites in the pentose-phosphate pathway (10). While Loricrin is a cell envelope protein. During epidermal cell cornification, layers of covalently cross-linked proteins on the cytoplasmic side of the plasma membrane form the cell envelope. It appears that loricrin is found on the inner surface of purified cell envelopes and hence is thought to be a major component of cell envelopes (8). This protein was found to be a substrate for transglutaminase (by Roop D.R. et al and (14)), it contains a very high content of glycine and serine residues, which account for 77% of the total. Tyrosine residues are interspaced. Though this protein does not have a high content of serine and tyrosine residues, since this protein contains some residues, and since it is on the cell inner surface, it is possible that it could be a substrate for protein kinases. ACMPK is a Ca++ dependent multifunctional protein kinase and can phosphorylate a wide variety of proteins, especially those containing a high concentration of serine and tyrosine residues. Hence it is probable that these two proteins loricrin and transaldolase are phosphorylated by ACMPK. Phosphorylation screening allows us to isolate only those substrates that are phosphorylated and hence the proteins that are matched in the database are probably substrates for protein kinases. Though we did not manage to sequence the full length of the cDNA as seen from the difference in sizes on the gel after plasmid rescue (fig 6) and the size of the DNA sequence (fig 7) the proteins matching the sequences are probably substrates for protein kinases.
In conclusion we can say that phosphorylation screening using a Ca++ dependent protein kinase ACMPK is a good method to elucidate probable substrates for protein kinases. And in the present paper it was shown that the proteins that matched the sequences were possible substrates for Ca++ dependent protein kinases though more sequencing needs to be done.
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Fig 1
Map of lZAP cloning vector
Fig 2
First screening at 20,000pfu/ml. Plate to the left is the plate without enzyme from which we cored our plaque. Plate to the right contained enzyme (ACMPK), it gives the background phosphorylation sites.
Fig 3
Second screening at 1000pfu/ml. Plate to the left is the plate without enzyme from which we cored the plaques, all the plaques underwent PCR and A was found to be the best for rescue of the DNA. Plate to the right contained enzyme (ACMPK), it gives the background phosphorylation sites.
Lanes 1 2 3 4 5 6 7 8 9 10
Figure 4
Gel electrophoresis of PCR products. Lanes 1 to 4 is of group G. Lane 5 is the control. Lane 6, 7, 8, 9 are samples H1 through H4 corresponding to plaques A, B, C, and D from the second screening. Lane 10 is the size marker.
Lanes 1 2 3 4 5 6 7 8 9 10 11
Figure 5
Gel of PCR products run by Wei Wei on 10/26/99. From left to right samples are:
Lane 1 standards; lane 2 positive control; lane 3 A3b; lane 4 B1E; lane 5 C1; lane 6 Da; lane 7 E1c; lane 8, F2C, lane 9, G1; lane 10 H1; lane 11, positive control.
Lanes 1 2 3 4 56 7 8 9 10
Figure 6
Gel electrophoresis after plasmid rescue. From left to right samples are: Lane 1--sampleH1 1, Lane 2--sampleH1 2 Lane 3--sampleH1 3 Lane 4--sampleH1 4
Figure 7A
The cDNA sequence (f2) is shown together with the predicted amino acid sequences
Figure 7B
The cDNA sequence (a3) is shown together with the predicted amino acid sequences.
Figure 7C
Protein sequence which matches with a expect of 7e-27 to cDNA sequence in figure 7A
Figure 7DProtein sequence which matches with an expect of 5e-24 to cDNA sequence in figure 7B.
Lanes 1 2 3 4 5 6 7 8 9 10 11 12
Figure 8
Gel for restriction mapping: Lane 7 corresponds to pBluescript, lane 8 corresponds to Pst and shows a band indicating the presence of a pst restriction site, lane 9 shows the presence of an E corI restriction site (where our cDNA was inserted), lanes 10, 11 and 12 show the presence of a XbaI site, Sal1 and BamH1 sites respectively.
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