Sequence similarity between myospryn and AKAP12
The muscle-specific protein, myospryn harbors a tripartite motif (TRIM) (19
) located in the carboxy-terminal 550 amino acids of the full-length protein (3,739 amino acids) leaving well over 3,000 amino acids of the protein uncharacterized. In an effort to gain insight into the potential function of myospryn in muscle we searched various protein motif databases for further structural information on the protein. One such protein family classification algorithm (Panther, Celera Genomics) revealed similarity between myospryn and AKAP12 (data not shown). AKAP12 is a member of the protein kinase A anchoring protein (AKAP) superfamily which bind to the regulatory subunits of protein kinase A (PKA) thereby acting as downstream scaffolds for the cAMP signaling pathway (10
). AKAP12, also known as gravin/AKAP250 and SSeCKS (Src-suppressed C kinase substrate), the human and mouse homologues, respectively, is a ubiquitously expressed, cytoplasmic scaffold for PKA (19
). As shown in , myospryn (amino acids 1,428 - 3,030) and gravin (amino acids 216 - 1,755) exhibit 18% identity and 35% similarity within a region spanning 1,500 amino acids. The modest similarity of myospryn to this AKAP prompted us to test for an interaction with PKA.
Figure 1 Sequence similarity between Myospryn and Gravin. A, Bioinformatics analysis revealed that Myospryn (amino acids 1,428 - 3,030) shares 18% amino acid identity and 35% positive similarity with the PKA anchoring protein Gravin/AKAP12 (amino acids 216 - 1,755). (more ...)
Myospryn interacts with the type II regulatory subunit of PKA
To establish that myospryn binds to PKA we co-expressed epitope tagged Myc-myospryn and individual FLAG-PKA subunits in COS cells and tested for interaction by co-immunoprecipitation (co-IP). For these experiments we initially tested the type II regulatory (R) subunit of PKA, RIIα, since it binds to most of the known AKAPs (22
). Total protein extracts from transfected cells were incubated in the presence of anti-Myc antibody and protein G sepharose beads and the immunoprecipitations were subjected to Western analysis using an anti-FLAG antibody. As shown in , these immunoprecipitations revealed that full-length myospryn is capable of interacting with the RIIα subunit of PKA in COS cells. pcDNA3-Myc vector and Myc-SSeCKS served as negative and positive controls respectively for RIIα binding. As an initial step for locating the interaction region we tested the ability of two different myospryn constructs, an amino-terminal myospryn construct (amino acids 1,370 - 3,096) and a carboxy-terminal fragment (amino acids 2,965 - 3,739), to interact with RIIα. The amino-terminal myospryn fragment did not immunoprecipitate with RIIα whereas the carboxy-terminal Spe fragment of myospryn interacted effectively with RIIα (). The above results are the first demonstration that myospryn interacts with PKA in a region encompassing amino acids 3,096 and 3,739.
To verify the above association myospryn and RIIα were subjected to GST pulldown experiments. Recombinant GST-RIIα coupled to glutathione sepharose beads effectively precipitated the myospryn carboxy-terminal fragment (). This interaction was specific since GST alone was unable to immunoprecipitate myospryn. Taken together, the mammalian cell co-IP and the GST pulldown assays confirm that myospryn and PKA interact under various experimental conditions.
The AKAP superfamily is generally categorized into two classes: 1) members which bind to a single type of regulatory (R) subunit (single-specificity) or 2) members that bind to both the type I and type II regulatory subunits (dual-specificity) (10
). Therefore, we tested whether myospryn could bind to additional R subunits of PKA using co-IP as described above. The remaining PKA regulatory subunits, type I (RIα and RIβ) and type II (RIIβ) were cloned into FLAG-tagged expression vectors and co-transfected individually along with the carboxy-terminal Myc-tagged Spe fragment in COS cells. As shown in , the carboxy-terminal region of myospryn was unable to interact with the other PKA regulatory subunits. Thus myospryn, like AKAP12, interacts specifically with the RIIα subunit of PKA and can thus be considered a type II single-specificity anchoring protein for PKA.
Myospryn and RII α interact in intact cells
To demonstrate that myospryn and RIIα interact in a cellular environment we tested whether a nuclear localized form of RIIα could recruit myospryn to the nucleus. Myospryn and RIIα are typically distributed in the cytoplasm when expressed individually in COS cells (, upper left panel and , middle lower panel). Therefore, for these experiments we designed an RIIα expression vector harboring a strong nuclear localization signal (NLS). As shown in , RIIα which normally resides in the cytoplasm (upper panels) is directed to the nucleus as a NLS-RIIα fusion protein (lower panels). When the NLS-RIIα expression vector was used in co-transfection experiments, RIIα could effectively target myospryn to the nucleus (, compare upper left and middle panels to lower middle panel).
Figure 2 Myospryn and RIIα interact in a cellular environment. A, RIIα and NLS-RIIα were transfected into COS cells and immunostained using an anti-RII antibody. RIIα exhibits a cytoplasmic/perinuclear localization whereas NLS-RIIα (more ...)
We also tested for an endogenous interaction between the two proteins. Myospryn antibodies are unable to immunoprecipitate native myospryn from muscle under standard conditions precluding our ability to analyze endogenous complex formation. Instead, we transfected a Myc-myospryn construct in COS cells and tested its ability to precipitate endogenous PKA. As shown in , Myc-myospryn immunoprecipitated endogenous PKA from COS cells whereas overexpression of pCNDA3-Myc alone was unable to do so. The above results reinforce the notion of a myospryn-PKA interaction in mammalian cells.
Myospryn and RII α colocalize in striated muscle
AKAPs are found at specific subcellular compartments which serves to direct PKA enzymatic activity in close proximity to its target substrate(s). Previously published reports described a Z-disc pattern of localization for RIIα in striated muscle (23
). In addition, it has been shown that myospryn localizes to the costamere at the periphery of myofibrils in periodic register with the Z-disc (17
). To determine whether myospryn and RIIα co-localize at the costameric region in striated muscle we performed double-label immunohistochemistry on adult mouse skeletal muscle longitudinal sections with antibodies directed against myospryn (17
) and the type II regulatory subunit (Upstate Biotechnology). As demonstrated in our previous study, myospryn exhibits a periodic staining pattern in longitudinal sections reflecting localization that is in register with the Z-disc (, upper panel). Similarly, the RII - specific antibodies exhibited a periodic staining pattern (, middle panel) consistent with previous data. When these images were superimposed the fluorescent label turned yellow specifically at the Z-disc indicative of co-localization (, bottom panel). In a complementary set of experiments we performed double label immunohistochemistry on transverse skeletal muscle sections. As shown in , myospryn and RII are co-localized along the periphery of muscle fibers. These results revealed that myospryn and the PKA RII
subunit are present in the same subcellular compartment in striated muscle in vivo
, reinforcing the notion that myospryn localizes PKA signaling to the peripheral Z-disc region of striated muscle.
Figure 3 Co-localization of Myospryn and RIIα in striated muscle. A, Double immunohistochemistry on longitudinal sections from hindlimb muscle. Upper panel, muscle sections using anti-Myospryn antibodies shows a striated pattern of expression (red) as (more ...)
Identification of three amphipathic helix PKA binding motifs in myospryn
Given the ability of the carboxy-terminal residues of myospryn (amino acids 3,096 - 3,739) to bind RIIα, we set out to identify the minimal PKA interaction domain within this region. It is worth noting that the PKA docking site in gravin is embedded within the region of similarity between the proteins yet this homologous region on myospryn did not bind to PKA. This is not entirely surprising since few AKAPs exhibit primary amino acid sequence conservation within their PKA-anchoring domains (10
). More importantly, it is the ability of a stretch of 14-18 amino acids to form an amphipathic helix that functions as a high affinity docking site for the R subunits of PKA (10
). Since we identified a large number of predicted α-helices in the carboxy-terminal region of myospryn we set out to functionally map the minimal interaction domain between myospryn and RIIα, using co-IP in COS cells.
We generated numerous amino-terminal, carboxy-terminal, and internal deletion constructs of the Spe fragment (amino acids 2,965 - 3,739) of myospryn and tested each one for its ability to bind RIIα. As shown in , three progressive carboxy-terminal deletion constructs in the context of the myospryn Spe fragment, designated Δ1, Δ2, and Δ3, respectively, interacted with RIIα. However, an additional deletion construct, CYTC, (amino acids 2,965 - 3,187) was unable to immunoprecipitate RIIα demonstrating the existence of a potential PKA docking site between amino acid 3,187 and 3,215 of myospryn. Upon examining the primary amino acid sequence between residues 3,187 and 3,215 we identified a 17-amino acid sequence, CKSLVSEMDKALDIHKD, predicted to form an amphipathic helix when subjected to helical wheel analysis (www.kael.net/helical_old.htm
) (, left, designated H1). To demonstrate that interaction with RIIα is occurring through the hydrophobic region of the predicted helix H1 a mutant construct was generated consisting of a single amino acid substitution of a leucine residue with a proline (L3197P) in the context of the Δ3. Introduction of a proline within the hydrophobic face of the helix is expected to disrupt the helix and interfere with the ability of myospryn to interact with RIIα. As predicted, the Δ3 - L3197P mutant construct was unable to interact with RIIα (, left panel). These results demonstrate the presence of a minimal amphipathic helix anchoring domain for PKA in myospryn.
Figure 4 Mapping of the RIIα-binding site of Myospryn. A, Coimmunoprecipitation demonstrating the identification of an RIIα interaction domain. Upper panel, Progressive Myc-tagged C-terminal myospryn deletions were cotransfected with FLAG-tagged (more ...)
We continued our mapping analysis testing various fragments within the COOH-terminal region of myospryn for interaction with PKA because of two unexpected findings. First, a deletion construct ΔN8.2 lacking the amino-terminal 250 amino acids of the myospryn Spe fragment (amino acids 3,216 - 3,739), which removes the aforementioned PKA-anchoring domain H1, interacted with RIIα (). Second, an internal deletion within the Δ2 construct, lacking the H1 anchoring domain, also interacted with RIIα suggesting the presence of an additional PKA anchoring domain between residues 3,216 and 3,340. Therefore, we generated a construct which overlapped with and included most of these residues (amino acids 3,267 - 3,403). When tested by co-IP, this region alone interacted with RIIα (, middle panel). Examination of the primary amino acid sequence in this construct revealed an 18-amino acid sequence, SMDTAKDTLETIVREAGE (amino acids 3,301 - 3,318), that is predicted to form an amphipathic helix (, middle, designated H2). Substitution of leucine in position 3,309 with a proline (L3309P) completely disrupted interaction with RIIα (, middle panel). These results demonstrate the existence of a second amphipathic helix H2 in myospryn.
To test whether eliminating both docking sites abolished interaction between myospryn and PKA, a construct was generated which removed both amphipathic helices H1 and H2 in the context of the Spe fragment (Δ3,187 - 3,375). Surprisingly, this construct effectively immunoprecipitated RIIα ( and data not shown) suggesting the existence of a third docking site for PKA between amino acids 3,375 and 3,739. To confirm this finding two additional amino-terminal deletions were generated, a construct consisting of amino acids 3,375 through 3,739 and a construct from amino acids 3,474 - 3,739. Each construct was tested for its ability to interact with RIIα. The co-IP experiments revealed that construct 3,375 - 3,739 interacted with RIIα whereas construct 3,474 - 3,739 did not () indicating a third anchoring site between amino acids 3,375 and 3,474. Since a minimal construct coding for amino acids 3,375 through 3,474 failed to express, a larger construct was generated extending from amino acids 3,375 to 3,565 and tested for interaction with RIIα. This fragment was sufficient to immunoprecipitate RIIα (). Examination of this region uncovered a predicted amphipathic helix between amino acids 3,421 and 3,437, EINELVEEYRLTVKESC, (, right, designated H3). Substitution of cysteine at position 3,437 with a proline in a construct expressing amino acids 3,375 - 3,565 (C3437P), which harbors amphipathic helix H3, severely disrupted the interaction with RIIα (, right panel). As summarized in the schematic , the above results confirm the existence of three bona-fide PKA-anchoring domains in myospryn each predicted to form an amphipathic helix and independently capable of interacting with RIIα.
Evolutionary conservation of the PKA-anchoring region in myospryn
To determine whether the PKA-anchoring function in myospryn has been evolutionarily conserved we compared the carboxy-terminal amino acid sequences of the mouse, human, and zebrafish orthologs of myospryn. Indeed, we identified all three amphipathic helices to be evolutionarily conserved in these three species with helix H3 exhibiting the highest degree of conservation (). To test whether zebrafish myospryn is capable of interacting with RIIα we amplified the carboxy-terminal sequences from a 96hr post-fertilization zebrafish embryo cDNA library and cloned this fragment into pcDNA3-Myc. We then tested for interaction with RIIα using coIP in COS cells. As shown in , zebrafish myospryn interacted effectively with RIIα clearly demonstrating conservation of AKAP function in myospryn.
Figure 6 A, Sequence alignment of the three PKA anchoring motifs. The C-terminal myospryn fragment Spe possesses significant sequence homology to the Zebrafish (Danio rerio) myospryn ortholog (amino acids 894 and 1161). This region in zebrafish myospryn harbors (more ...)
Myospryn is an AKAP that is phoshorylated by PKA in vitro
Given that some AKAPs can be regulated by PKA phosphorylation we searched the myospryn open reading frame for potential PKA phosphorylation sites using the Motif Scan program (scansite.mit.edu
). This analysis revealed three sequences located in the amino-terminal region of the protein that conform to the consensus PKA recognition sequence, KRXS () (26
). A FLAG-tagged amino-terminal construct (amino acids 73 -743) harboring these sites, KRGS142
, was expressed in COS cells, immunoprecipitated with anti-FLAG antibodies and subsequently used as a substrate in an in vitro
kinase assay with recombinant PKA catalytic subunit (New England Biolabs) and γ-32
P-ATP (Perkin Elmer). As shown in , this construct was phosphorylated by recombinant PKA (left lane). As a negative control, a pcDNA3-FLAG vector transfected in COS cells and immunoprecipited with anti-FLAG was not phosphorylated by PKA (data not shown).
Figure 7 Myospryn is a substrate for PKA. A, Schematic of the N-terminal fragment of myospryn (amino acids 73-743) shows three consensus PKA phosphorylation sites between amino acids 138-158. B, The putative PKA phosphorylation sites are phosphorylated by PKA (more ...)
To further demonstrate specificity of PKA phosphorylation at these particular sites a myospryn construct lacking these sequences (amino acids 138 - 158) was expressed in COS cells, immunoprecipitated and subjected to an in vitro kinase assay. As shown in , this deletion construct was unable to be phosphorylated by PKA demonstrating the specificity of phosphorylation at one or all three of these sites (right lane). Moreover, subjecting these reactions to the PKA peptide inhibitor, PKI (Promega), completely blocked phosphorylation on myospryn (, compare right lane to middle lane). An additional negative control reaction incubated, under the same conditions, in the absence of recombinant PKA was unable to phosphorylate myospryn (, left lane). These results show that, in addition to anchoring PKA, myospryn has the potential to serve as a substrate for the cAMP signaling pathway in muscle.