Goodpasture antigen-binding protein (GPBP) directs myofibril formation: identification of intracellular downstream effector 130-kDa GPBP-interacting protein (GIP130)

Abstract

Goodpasture antigen-binding protein-1 (GPBP-1) is an exportable non-conventional Ser/Thr kinase that regulates glomerular basement membrane collagen organization. Here we provide evidence that GPBP-1 accumulates in the cytoplasm of differentiating mouse myoblasts prior to myosin synthesis. Myoblasts deficient in GPBP-1 display defective myofibril formation, whereas myofibrils assemble with enhanced efficiency in those overexpressing GPBP-1. We also show that GPBP-1 targets the previously unidentified GIP130 (GPBP-interacting protein of 130 kDa), which binds to myosin and promotes its myofibrillar assembly. This report reveals that GPBP-1 directs myofibril formation, an observation that expands its reported role in supramolecular organization of structural proteins to the intracellular compartment.

FIGURE 1.

Col4a3bp expression in differentiating C2C12 myoblasts. A, C2C12 cells were serum-deprived, and at the indicated days, 50 μg of cell lysates were subjected to Western blot analysis with antibodies targeting MyoHC (Myosin), GPBP-1 and -2 (mAb 14), GPBP-1 and -3 (mAb e26), and tubulin. The bracketed arrows and arrowheads denote the position of GPBP polypeptides of the indicated molecular mass. B, the protein expression of mGPBP-1 in C2C12 differentiation assays (n = 2) was assessed by Western blot and quantified by densitometry with respect to tubulin expression. C, the RNA extracted from the cells in A was used for quantitative RT-PCR to determine the relative expression of the indicated mRNAs (bars). B and C, values are in -fold expression with respect to undifferentiated cells (day 0), which was set at 1. D, 3-day differentiated C2C12 cells were subjected to subcellular fractionation, and 15 μg of each of the indicated fractions were analyzed by Western blot for specific mGPBP-1 detection with mAb e26. Error bars, S.D.

FIGURE 2.

Col4a3bp expression regulates myogenesis in C2C12 myoblasts. A, C2C12 cells were subjected to a 3-day differentiation and analyzed by confocal microscopy using rabbit polyclonal antibodies specific for Col4a3bp products and a mouse mAb for MyoHC detection. The white arrows indicate cells with increased intracellular expression of mGPBP-1 in which MyoHC could not be detected. Bars, 60 μm. B, C2C12 cells were transfected with construct pSi-GPBP/GPBPΔ26–2, and the indicated clones were analyzed either for Col4a3bp (mAb 14) or tubulin expression using Western blot procedures (left) or subjected to a 3-day differentiation and MyoHC expression analyzed as in A using conventional immunofluorescence microscopy (right). Bars, 30 μm. C, C2C12 cells were differentiated for 3 days, and mGPBP-1 and MyoHC expression was analyzed as in A. The arrow and arrowhead pinpoint mGPBP-1 aggregates in more and less differentiated cells, respectively. Bars, 20 μm. In this and following figures, nuclei are DAPI-stained and displayed in blue.

FIGURE 3.

Myoblast cell lines derived from gpbp-1^−/− mice show impaired differentiation. A, 50 μg of lysates from gpbp-1^+/+ (+/+) or gpbp-1^−/− (−/−) myoblasts were either analyzed by Western blot for mGPBP-1 and -3 (mAb e26) and tubulin detection or dephosphorylated with λ-phosphatase and similarly analyzed to detect mGPBP-1 and -2 (mAb 14). B, the cells in A were subjected to RNA extraction, RT, and conventional (left) or quantitative (right) PCR to assess levels of the indicated mRNAs. The products of the conventional PCR were analyzed by agarose gel electrophoresis and stained with ethidium bromide. Numbers and bars indicate the size (bp) and position of the standard DNA markers, and the arrowheads denote the position of the indicated cDNA products. The plot reflects the relative expression of the indicated mRNA as -fold level of gpbp-1^−/− (−/−) with respect to gpbp-1^+/+ (+/+), which was set at 1. C, myoblasts from gpbp-1^+/+ (+/+) and gpbp-1^−/− (−/−) mice were differentiated for 5 days and analyzed by phase-contrast microscopy. Original magnification was ×100. D, the cultures in C were stained with antibodies for MyoHC (green), and individual representative cells were analyzed by confocal microscopy. Inset, characteristic striation of myofibrils assembled in differentiated gpbp-1^+/+ (+/+) cells. Bars, 20 μm. E, the cells in C were analyzed by EM to visualize myofibrillar material. Note the contrast between myofibrillar material in gpbp-1^+/+ myoblasts, which displayed well developed sarcomeric organization and myofibrillar material in gpbp-1^−/− myoblasts lacking sarcomeric organization. F, micrograph showing a cytoplasmic body characteristic of gpbp-1^−/− myoblast cultures (left) and a magnified cytoplasmic body displaying radial distribution of myofibrillar material (right). G, 50 μg of lysates from undifferentiated (U) or 5-day differentiated (D) gpbp-1^+/+ (+/+) or gpbp-1^−/− (−/−) myoblasts were subjected to Western blot analysis with antibodies against the indicated proteins. H, 30,000 myoblasts from gpbp-1^+/+ (+/+) (n = 2) and gpbp-1^−/− (−/−) (n = 2) mice were cultured in growth medium, and cells were quantified at days 1, 2, and 3 after seeding. Division rates were estimated and represented as means ± S.E. (error bars) of -fold growth at the indicated times after day 1. Differences in growth rate between +/+ and −/− cultures lacked statistical significance.

FIGURE 4.

Myoblast cell lines derived from Tg-hGPBP-1 mice show accelerated differentiation. A, lysates of undifferentiated myoblasts derived from wild type (WT) or Tg-hGPBP-1 (Tg) mice were analyzed by Western blot with specific antibodies for GPBP (mAb N27) or for tubulin, used as loading control. The arrowhead indicates the position of reactive polypeptide present only in Tg-hGPBP-1 lysates representing FLAG-tagged GPBP-1. B, the lysates in A were subjected to immunoprecipitation (IP) and Western blot analysis of precipitates (WB) using the indicated antibodies. Numbers and bars in A and B denote the size (kDa) and position of individual molecular mass markers. C, lysates (50 μg) of undifferentiated (U) and 4-day differentiated (D) WT or Tg-hGPBP-1 myoblasts were analyzed by Western blot using antibodies specific for the indicated proteins. D, the cells in A were differentiated for 2 days and analyzed by phase-contrast microscopy. Original magnification was ×100. E, representative confocal microscopy images of the cultures in D stained with antibodies for MyoHC visualization (green). Bars, 20 μm. F, representative confocal microscopy images of a differentiated Tg-hGPBP-1 culture stained with MyoHC-specific (green) and GPBP-specific (red) antibodies. The arrowhead denotes GPBP-1 intimately associated with an A band in a striated myofibril. Bars, 5 μm. The graphs represent the distribution of fluorescence intensity in the region indicated by the arrow in a nascent myofibril. The arrowheads denote co-localization of the indicated proteins.

FIGURE 5.

GIP130 is a FILIP1L isoform having features of a structural protein that is preferentially expressed in striated muscle. A, the primary structure of GIP130 is shown in one-letter code. The predicted coiled-coiled region and canonical leucine zippers are highlighted with gray and black backgrounds, respectively. B, shown are the exon-intron structures of pre-mRNA of GIP130 (GenBank^TM accession number AF514867) and FILIP1L-102 (GenBank^TM accession BC027860) transcribed from FILIP1L at 3q12.1 and exon sequence of I20. C, ³²P-labeled cDNAs representing both GIP130 and FILIP1L-102 (I20 probe) or GIP130 (exon I–IV probe) or β-actin mRNAs were used to subsequently probe a premade Northern blot of poly(A⁺) RNA from the indicated human tissues. Shown are 5-day (I20), 8-day (exons I–IV), and 0.5-day (β-actin) exposures at −70 °C, respectively. The arrowheads and numbers indicate the positions and sizes (kb) of identified mRNA species, whereas the bars and numbers similarly denote RNA markers. D, lysates (50 μg) of HEK 293 cells transfected with either pRK-c-Myc-GIP130 (GIP130), pRK5-c-Myc-FILIP1L-102 (FILIP1L-102), or pRK5-c-Myc vector (control) were analyzed by Western blot with the indicated antibodies. E, schematic alignment of primary structures (bars) of GIP130 and FILIP1L-102 polypeptides expressed in D. Gray shading and black boxes represent coiled-coils and leucine zippers, respectively.

FIGURE 6.

GPBP binds and phosphorylates GIP130, a previously unrecognized myofibrillar component. A, 200 ng of yeast FLAG-tagged rGPBP-1 and/or rGIP130 were subjected to phosphorylation and mixtures analyzed by Western blot and autoradiography (³²P) during 3 and 16 h and subsequently probed with FLAG-specific antibodies (anti-FLAG). The empty and filled arrowheads denote the positions of rGIP130 and rGPBP-1 primary products, respectively. Numbers and bars at the left indicate size (kDa) and position of individual molecular mass markers. B, HEK 293 cells expressing rGPBP-1 (control) or rGPBP-1- and c-Myc-tagged rGIP130 (GIP130) were lysed and subjected either to Western blot analysis with the indicated antibodies (Lysates) or to immunoprecipitation with anti-c-Myc affinity resin and similarly analyzed (IP). C, protein extracts of mouse diaphragm (50 μg) were analyzed by Western blot (Input) or subjected to immunoprecipitation (500 μg) in the absence (−) or presence of mAb N27 (N27) and similarly analyzed with the indicated antibodies. In B and C, primary and related products are denoted by arrows and their size (kDa). D, myofibrils isolated from mouse diaphragm were analyzed by confocal microscopy for detection of the indicated proteins using rabbit polyclonal antibodies (Myosin), chicken polyclonal antibodies (GPBP-1), and mAb3_GIP (GIP). The graphs represent the distribution of fluorescence intensities in the region indicated by the arrow in the merged image. The arrowheads denote co-localization of the three proteins at the M lines of A bands.

FIGURE 7.

GIP130 binds to myosin and promotes its myofibrillar assembly in differentiating C2C12 cells. A, 2 μg of mouse myofibril protein extract (lanes 2) and 200 ng of BSA (lanes 3) were subjected to SDS-PAGE along with suitable molecular mass markers (lanes 1) and either stained (Coomassie) or transferred to a PVDF membrane and probed with either FLAG-tagged rGIP130 (1 μg/ml) and mAb3_GIP antibodies (far Western), or with mAb3_GIP antibodies (control). At the left of the composite are the size (kDa) and identities of molecular mass markers: rabbit skeletal muscle MyoHC (Myosin), phosphorylase b (PYGB), bovine serum albumin (BSA), and ovalbumin (OVA). B, myofibril (MF) protein extracts used in A (500 μg) were incubated with (+) or without (−) FLAG-tagged rGIP130 (GIP130), and mixtures were subjected to immunoprecipitation using anti-FLAG antibodies. Precipitates were analyzed by Western blot with suitable antibodies for MyoHC (Myosin) or GIP130 (GIP) detection. IgG heavy chain is shown as a loading control (antibody). C, C2C12 (control) or C2C12 expressing rGIP130 (GIP) cells were subjected to 3-day differentiation. Undifferentiated (U) or differentiated (D) cells were used for cytoskeleton extraction, and supernatants (S) and pellets (P) were analyzed by Western blot using antibodies specific for the indicated proteins. D, the RNA from undifferentiated or 3-day differentiated C2C12 cells was extracted, and the indicated mRNAs were selectively amplified by RT-PCR. Amplification mixtures in the absence (−) or presence of undifferentiated (U) or differentiated (D) RNA were analyzed by agarose gel electrophoresis and stained with ethidium bromide. Indicated are the size and position of DNA markers. E, C2C12 cells were transfected with pSi-mGIP-2 to yield clones differing in mGIP130 expression as assessed by quantitative RT-PCR. Shown are the relative mGIP130 expressions of two sh-mGIP clones (4 and 6) with respect to a clone generated with the corresponding empty vector (control). Shown are the means ± S.D. (error bars). F, the C2C12 clones in E were differentiated for 3 days and analyzed by confocal microscopy for myosin detection. Inset, characteristic striation of myofibrils assembled in differentiated control clone. In contrast, cells of clone 6 displayed loose myofibrils without striation, and cells of clone 4 lacked myofibrils. Bars, 10 μm.

FIGURE 8.

GIP130 co-localizes with GPBP-1 and myosin during differentiation. C2C12 myoblasts were co-transfected with individual constructs expressing rGPBP-1 or rGIP130 and then subjected to differentiation. After 1 day (top row) or 3 days (bottom row) of differentiation, cultures were analyzed by confocal microscopy to visualize the indicated proteins. The graphs represent the distribution of fluorescence (cyan and green) intensity in the region indicated by the arrow in the inset of the merged images (top row, left graphs and bottom row, right graphs). The arrowheads denote co-localization of the indicated proteins. Bars, 10 μm.

FIGURE 9.

GPBP-1 promotes the myofibrillar assembly of GIP130 and myosin. A, gpbp-1^+/+ and gpbp-1^−/− myoblasts were transfected with GIP130-EGFP-expressing construct, differentiated for 3 days, and analyzed by confocal microscopy to visualize the indicated proteins. B, gpbp-1^−/− myoblasts were transfected with the indicated constructs, and cultures were differentiated for 3 days and analyzed by confocal microscopy for detection of the indicated proteins. In A and B, myosin was detected with MyoHC-specific antibodies, and GIP130-EGFP and EGFP were detected by autofluorescence. In B, GPBP-1 was detected with anti-GPBP-specific antibodies under conditions in which endogenous mGPBP-2 was not detectable Bars, 10 μm.