Using substrate-binding variants of the cAMP-dependent protein kinase to identify novel targets and a kinase domain important for substrate interactions in Saccharomyces cerevisiae - PubMed (original) (raw)

Using substrate-binding variants of the cAMP-dependent protein kinase to identify novel targets and a kinase domain important for substrate interactions in Saccharomyces cerevisiae

Stephen J Deminoff et al. Genetics. 2006 Aug.

Abstract

Protein kinases mediate much of the signal transduction in eukaryotic cells and defects in kinase function are associated with a variety of human diseases. To understand and correct these defects, we will need to identify the physiologically relevant substrates of these enzymes. The work presented here describes a novel approach to this identification process for the cAMP-dependent protein kinase (PKA) in Saccharomyces cerevisiae. This approach takes advantage of two catalytically inactive PKA variants, Tpk1K336A/H338A and Tpk1R324A, that exhibit a stable binding to their substrates. Most protein kinases, including the wild-type PKA, associate with substrates with a relatively low affinity. The binding observed here was specific to substrates and was dependent upon PKA residues known to be important for interactions with peptide substrates. The general utility of this approach was demonstrated by the ability to identify both previously described and novel PKA substrates in S. cerevisiae. Interestingly, the positions of the residues altered in these variants implicated a particular region within the PKA kinase domain, corresponding to subdomain XI, in the binding and/or release of protein substrates. Moreover, the high conservation of the residues altered and, in particular, the invariant nature of the R324 position suggest that this approach might be generally applicable to other protein kinases.

PubMed Disclaimer

Figures

Figure 1.—

The Tpk1KH variant, but not the wild-type Tpk1, was physically associated with known PKA substrates. (A) The Tpk1KH variant was detected in immunoprecipitates with two PKA substrates, Cdc25 and Cki1. An HA epitope-tagged version of either the wild-type Tpk1 or the Tpk1KH variant was precipitated from cell extracts with an anti-HA affinity matrix. The bound substrate was eluted, separated on an SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. The relative amount of substrate present was then assessed by Western immunoblotting with antibodies specific for the myc epitope on the particular substrate present, either Cdc25 or Cki1. Three different amounts of the final immunoprecipitates and original inputs were loaded onto the gels, corresponding to 1, 3, and 10 μl of the 30 and 1000 μl present, respectively. (B) A Western immunoblot showing the relative levels of the wild-type Tpk1 and Tpk1KH present in the immunoprecipitates shown in A. (C) The Tpk1KH and Tpk1R324A variants were defective for PKA kinase activity in vitro. A myc epitope-tagged Cdc25 protein was incubated with [γ-32P]ATP and the wild-type Tpk1, Tpk1KH, or Tpk1R324A for 30 min at 25°. The reaction products were separated on an SDS-polyacrylamide gel and the extent of phosphorylation was assessed by autoradiography. (Bottom) A Western immunoblot showing the relative levels of the Tpk1 enzymes present in the kinase reactions. (D) The overexpression of the wild-type Tpk1 resulted in a growth defect in yeast cells. Wild-type cells were transformed with plasmids expressing the wild-type Tpk1, Tpk1KH, or Tpk1R324A from the CUP1 promoter. The cells were then plated to YM minimal media containing either 0 (Uninduced) or 100 μM (Induced) CuSO4 and incubated at 30° for 3 days.

Figure 2.—

The interactions with the Tpk1KH variant were specific to PKA substrates. (A) The Tpk1KH variant did not associate with a fragment of Cki1 that lacks the sites of PKA phosphorylation. The relative amount of three different proteins, Cdc25 (codons 51–330), Cki1 (2–200), and Cki1-N (274–471), precipitating with the Tpk1KH variant was assessed as described in Figure 1A. The asterisk denotes a nonspecific protein band recognized by the α-myc epitope antibody. (B) A two-hybrid assay showing that the Tpk1KH variant interacted with the known substrates, Cdc25 and Cki1, but not with the nonsubstrates, Rim15-N and Toa2. The wild-type Tpk1 failed to interact with any of these test proteins. For these assays, the Tpk1 fusions to the Gal4 activation domain were under the control of the inducible promoter from the CUP1 gene (see

materials and methods

). Rim15-N (codons 361–657) was a negative control that was not phosphorylated by PKA. (C) A Western immunoblot showing the relative levels of the wild-type Tpk1 and Tpk1KH present in the two-hybrid strains shown in B. (D) Toa2 was not phosphorylated by PKA in vitro. Protein A fusions to Toa2 and a portion of Cdc25 (codons 51–330) were precipitated from yeast cell extracts and incubated with PKA and [γ-32P]ATP. The reaction products were separated on an SDS-polyacrylamide gel and the level of phosphorylation was assessed by autoradiography. (Right) A Western immunoblot showing the relative levels of the two substrates present in the kinase assays. (E) The Tpk1KH variant interacted with a Rim15 fragment containing the known sites of PKA phosphorylation (codons 1431–1671) but not with the Rim15-N fragment in a standard two-hybrid assay.

Figure 3.—

The Tpk1KH variant did not interact with proteins that were not substrates of PKA, including those that possessed sequences matching the PKA consensus phosphorylation site. (A) A two-hybrid assay examining the interactions between the Tpk1KH variant and a number of proteins that were not phosphorylated by PKA in vitro. Rim15 served as the positive control in these assays. (B) A Western immunoblot showing the relative levels of the different substrate proteins tested.

Figure 4.—

Tpk1KH binding to substrates required amino acid residues known to be important for the interaction between the wild-type PKA and its substrates. (A) A two-hybrid assay assessing the interactions between Tpk1KH variants and two substrates, Rim15 and Cdc25. The Tpk1KH (E → A) variant has replaced both E171 and E274 with an alanine residue. These glutamic acid residues have been shown previously to be important for Tpk1 interactions with peptide substrates (G

ibbs

and Z

oller

1991a; K

emp

et al. 1994). (B) A Western immunoblot showing the relative levels of the two Tpk1 enzymes present in the two-hybrid strains with Cdc25 as substrate. (C) A Yak1 fragment containing codons 243–361 is phosphorylated at a single site by PKA in vitro. Protein A fusions to Yak1 with either a wild-type (Yak1) or altered PKA site [Yak1(−)] were precipitated from yeast cell extracts and incubated with PKA and [γ-32P]ATP. The reaction products were separated on an SDS-polyacrylamide gel and the level of phosphorylation was assessed by autoradiography. (Right) A Western immunoblot showing the relative levels of the two Yak1 proteins present in the kinase assays. (D) A Yak1 fragment containing only a single site of PKA phosphorylation (codons 243–361) was recognized by the Tpk1KH variant in a two-hybrid assay.

Figure 5.—

Tpk1KH and Tpk1R324A were the only inactive variants of Tpk1 that interacted with known substrates of PKA. (A) A schematic representing the protein kinase domain of Tpk1 (aa 90–390). The Tpk1 residues altered here are shown in boldface type; the relative positions of these residues in Tpk1 are indicated by the numbers below the diagram. The roman numerals above the diagram indicate the Hanks and Hunter subdomain designations for the indicated regions of the protein kinase domain (H

anks

and H

unter

1995). (B) A two-hybrid analysis showing the interactions observed between different Tpk1 variants and four known PKA substrates, Rim15, Yak1, Cki1, and Cdc25. An interaction is indicated by growth on the YM minimal medium lacking adenine (Ade_−_). (C) Plates showing the relative two-hybrid interaction observed for the Tpk1KH and Tpk1R324A variants with the Rim15 substrate. (D) Western immunoblots showing the relative levels of the different Tpk1 variants and substrates present in the strains used for the two-hybrid analyses in B and C. Note that very low levels of the wild-type Tpk1 were found in these two-hybrid strains. (E) The substrates used in the above two-hybrid analysis were all efficient in vitro substrates for PKA. Each of the indicated substrate proteins was precipitated from yeast cell extracts and incubated with the yeast Tpk1 and [γ-32P]ATP as described in

materials and methods

. The reaction products were separated on an SDS-polyacrylamide gel and the level of phosphorylation was assessed by autoradiography.

Figure 6.—

The Dot6 protein was identified as a candidate substrate for PKA. (A) Dot6 interacted with the Tpk1KH variant in a two-hybrid assay. (B) A schematic showing the positions of the five PKA consensus phosphorylation sites in the Dot6 protein; the sites are indicated by stars. A single myb domain, a DNA-binding motif originally identified in the c-myb oncoprotein, is present at the N-terminus of this protein (S

inger

et al. 1998). (C) The Dot6 protein was phosphorylated in vitro by the bovine PKA (bPKA) and the yeast Tpk1. (D) The Dot6 protein was recognized by an antibody specific for phosphorylated PKA sites. GST–Dot6 and GST–Gcs1 fusion proteins were precipitated with an anti-GST antibody, separated on an SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. The phosphorylation state of these proteins was then assessed by Western immunoblotting with an anti-PKA substrate antibody (Cell Signaling) that recognizes the phosphorylated forms of PKA target sites. The accompanying blot with an anti-GST antibody shows the relative levels of each fusion protein. (E) Dot6 recognition by the anti-PKA substrate antibody. The immunoprecipitated GST–Dot6 fusion was treated either with λ phosphatase only (PPase) or λ phosphatase and then bovine PKA and ATP (PPase/PKA). The reaction products were separated by SDS–PAGE electrophoresis, blotted to a nitrocellulose membrane, and analyzed by Western immunoblotting with either the anti-PKA substrate or anti-GST antibodies.

References

1. Adams, J. A., 2001. Kinetic and catalytic mechanisms of protein kinases. Chem. Rev. 101: 2271–2290. - PubMed
1. Arnsten, A. F., B. P. Ramos, S. G. Birnbaum and J. R. Taylor, 2005. Protein kinase A as a therapeutic target for memory disorders: rationale and challenges. Trends Mol. Med. 11: 121–128. - PubMed
1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman et al., 1995. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
1. Broach, J. R., 1991. RAS genes in Saccharomyces cerevisiae: signal transduction in search of a pathway. Trends Genet. 7: 28–33. - PubMed
1. Budovskaya, Y. V., H. Hama, D. B. DeWald and P. K. Herman, 2002. The C terminus of the Vps34p phosphoinositide 3-kinase is necessary and sufficient for the interaction with the Vps15p protein kinase. J. Biol. Chem. 277: 287–294. - PubMed

Using substrate-binding variants of the cAMP-dependent protein kinase to identify novel targets and a kinase domain important for substrate interactions in Saccharomyces cerevisiae - PubMed (original) (raw)