A novel protein phosphatase is a binding partner for the protein kinase domains of UNC-89 (Obscurin) in Caenorhabditis elegans

Abstract

Mutation of the Caenorhabditis elegans gene unc-89 results in disorganization of muscle A-bands. unc-89 encodes a giant polypeptide (900 kDa) containing two protein kinase domains, PK1 and PK2. Yeast two-hybrid screening using a portion of UNC-89 including PK2, yielded SCPL-1 (small CTD phosphatase-like-1), which contains a C terminal domain (CTD) phosphatase type domain. In addition to the PK2 domain, interaction with SCPL-1 required the putative autoinhibitory sequence, and immunoglobulin (Ig) and fibronectin type 3 (Fn3) domains lying N-terminal of the kinase domain. SCPL-1 also interacts with PK1, and it similarly requires the kinase domain and upstream Fn3 and Ig domains. Analogous regions from the two other giant kinases of C. elegans, twitchin and TTN-1, failed to interact with SCPL-1. The interaction between SCPL-1 and either Ig-Fn3-PK2 or Fn3-Ig-PK1 was confirmed by biochemical methods. The scpl-1b promoter is expressed in the same set of muscles as unc-89. Antibodies to SCPL-1 localize to the M-line and a portion of the I-band. Bacterially expressed SCPL-1 proteins have phosphatase activity in vitro with properties similar to previously characterized members of the CTD phosphatase family. RNA interference knockdown results in a defect in the function of egg-laying muscles. These studies suggest a new role for the CTD phosphatase family, that is, in muscle giant kinase signaling.

Figure 1.

(A) Yeast two-hybrid assays demonstrate the specificity of the interaction of UNC-89 PK2 with SCPL-1. Left, schematic representation of baits used to test full-length SCPL-1a and -b prey. Right, images of yeast growth on −Ade plates. Note that for UNC-89, interaction requires the catalytic core (PK2) plus the N-terminal Ig and Fn3 domains, and the C-terminal autoinhibitory domain. Note that comparable regions from the two other giant kinases in the worm, twitchin and TTN-1, fail to interact with SCPL-1. (B) The PK1 region of UNC-89 also interacts with SCPL-1 in the yeast two-hybrid assay. Domain mapping indicates that interaction of the PK1 region with SCPL-1 requires, in addition to the catalytic core (PK1), the Fn3 and Ig domains. +, growth and −, no growth on −Ade plates. (C) Only the phosphatase domain of SCPL-1 is required for interaction to the PK regions of UNC-89 in yeast two-hybrid assays. The indicated portions of SCPL-1a and -1b were tested as bait against Fn3-Ig-PK1 (PK1) or Ig-Fn3-PK2 (PK2) prey. +, growth and −, no growth on −Ade plates. The colored bar indicates the minimal region of SCPL-1a/b required for interaction.

Figure 2.

Verification of interaction between SCPL-1 and UNC-89 PK1 or PK2 regions. (A) In the same yeast cell, myc-tagged SCPL-1b and individual HA-tagged derivatives of the UNC-89 PK2 region were expressed. 11/12, 11/13 … refer to the derivatives presented in Figure 1A. Total protein lysates were incubated with agarose beads coated with antibodies to the HA tag, washed, and eluted, and portions were run on two gels and blotted. One blot was reacted with anti-HA to detect the presence of the PK2 derivatives (top). As shown above, appropriately sized proteins were detected from each PK2 derivative. The other blot was reacted with anti-myc to detect the presence of SCPL-1 that might have been brought down with the PK2 protein (bottom). Only derivative 15/14 (Ig-Fn3-PK2-autoinhibitory region) is coimmunoprecipitated. An arrow designates the position on the blot of myc-SCPL-1b from the lysate. (B) Bacterially expressed MBP-SCPL-1a interacts with yeast expressed HA-PK1 or PK2. Total protein extracts were prepared from yeast expressing either HA-PK2 15/14 (see Figure 1A) or HA-PK1 AB (see Figure 1B), incubated with agarose beads coated with antibodies to HA, washed, incubated with purified, bacterially expressed MBP or MBP-SCPL-1a, and washed. The proteins were eluted, and portions of each sample were run on two gels and blotted. One blot was reacted with anti-HA to detect the presence of the yeast expressed protein, the other blot was reacted with anti-MBP to detect possible binding with MBP or MBP-SCPL-1a. (C) Coomassie stained gel of purified MBP-SCPL-1a and MBP-SCPL-1b. In each case, the top band is likely to be the full-length fusion protein; the second band may have resulted from degradation.

Figure 3.

The SCPL family of proteins in C. elegans and demonstration that UNC-89 PK2 region specifically interacts with SCPL-1. (A) Schematic representation of domain organization of proteins containing CTD type (or FCPH) phosphatase domains. C. elegans has five genes that encode proteins with FCPH domains: one domain called CeFCP-1 is more closely related to FCP proteins in that it has both phosphatase and BRCT domains. The others, including SCPL-1, which interacts with UNC-89, are more closely related to small CTD phosphatases (called SCPs), and they are designated SCP-L. The percentages indicate the percentage of identical amino acids in the ∼200-residue phosphatase domains, compared with the phosphatase in CeB0379.4a (SCPL-1). (B) By yeast two-hybrid assays, the UNC-89 PK2 region interacts with SCPL-1, but not SCPL-2, -3, or -4. The comparable regions of twitchin and TTN-1 fail to interact with any of the SCPL proteins. +, growth and −, no growth on −Ade plates.

Figure 4.

Expression pattern of the SCPL-1b promoter and characterization of antibodies to SCPL-1. (A) SCPL-1b is expressed in the same muscle cells as UNC-89. The exon/intron structure of the SCPL-1b gene is shown, as predicted on WormBase. 7.5 kb of DNA sequence upstream of the initiator methionine together with codons for a few amino acids of the first exon of SCPL-1b were fused in-frame with GFP, and used to create transgenic animals, and the sites of GFP expression were recorded. This promoter is expressed in pharyngeal (left), vulval (center), and body wall muscle (right). (B) Anti-SCPL-1b and anti-SCPL-1a antibodies specifically recognize SCPL-1b and SCPL-1a, respectively. Above is shown immunogens used to generate rabbit antibodies that were later affinity purified using the same regions. Below are Western blots reacted with the designated antibodies. In each case, the left-most three lanes are worm extracts, and the right-most lanes are yeast extracts. WT, wild type; scpl-1(ok1080) and scpl-1(gk283) are intragenic deletions of the scpl-1 gene. Vector, Myc-SCPL-1a, and Myc-SCPL-1b are yeast harboring either the empty vector or myc-tagged SCPL-1 isoforms. (C) Anti-SCPL-1 localize to M-lines and I-bands in body wall muscle. Anti-SCPL-1b and anti-UNC-89 were coincubated with wild-type worms, and the muscle was imaged by immunofluorescence microscopy. The images show a portion of one body wall muscle cell. Weak labeling of the M-line and a portion of the I-band is seen. UNC-89 is a marker of the M-line. Bar, 10 μm.

Figure 5.

Biochemical properties of SCPL-1. SCPL-1b was expressed as an MBP fusion protein in E. coli, and they are shown to have phosphatase activity toward a model substrate, p-nitrophenyl phosphate (A). Enzymatic properties were very similar to previously characterized FCPs and SCPs, including a pH optimum of ∼5.0 (B), preference for Mg⁺² (C), and unusual response to small-molecule inhibitors (not inhibited by NaF or Na₃VO₄ but inhibited by BeF₃⁻ and AlF₄; shown in D).