Isao Matsuura

Cyclin-dependent kinases regulate the antiproliferative function of Smads

Transforming growth factor-β (TGF-β) potently inhibits cell cycle progression at the G1 phase. Smad3 has a key function in mediating the TGF-β growth-inhibitory response. Here we show that Smad3 is a major physiological substrate of the G1 cyclin-dependent kinases CDK4 and CDK2. Except for the retinoblastoma protein family, Smad3 is the only CDK4 substrate demonstrated so far. We have mapped CDK4 and CDK2 phosphorylation sites to Thr 8, Thr 178 and Ser 212 in Smad3. Mutation of the CDK phosphorylation sites increases Smad3 transcriptional activity, leading to higher expression of the CDK inhibitor p15. Mutation of the CDK phosphorylation sites of Smad3 also increases its ability to downregulate the expression of c-myc. Using Smad3-/- mouse embryonic fibroblasts and other epithelial cell lines, we further show that Smad3 inhibits cell cycle progression from G1 to S phase and that mutation of the CDK phosphorylation sites in Smad3 increases this ability. Taken together, these findings indicate that CDK phosphorylation of Smad3 inhibits its transcriptional activity and antiproliferative function. Because cancer cells often contain high levels of CDK activity, diminishing Smad3 activity by CDK phosphorylation may contribute to tumorigenesis and TGF-β resistance in cancers.

Repression of Smad transcriptional activity by PIASy, an inhibitor of activated STAT

Smad proteins mediate transforming growth factor β (TGF-β)-inducible transcriptional responses. Protein inhibitor of activated signal transducer and activator of transcription (PIAS) represents a family of proteins that inhibits signal transducer and activator of transcription and also regulates other transcriptional responses. In an effort to identify Smad-interacting proteins by a yeast three-hybrid screen with Smad3 and Smad4 as baits, we identified PIASy, a member of the PIAS family. In yeast, PIASy interacts strongly with Smad4 and also with receptor-regulated Smads. In mammalian cells, PIASy binds most strongly with Smad3 and also associates with other receptor-regulated Smads and Smad4. The interaction between Smad3 and PIASy is increased in the presence of TGF-β and occurs through the C-terminal domain of Smad3. Moreover, Smad3, Smad4, and PIASy can form a ternary complex. PIASy does not inhibit Smad complex binding to DNA, but it represses Smad transcriptional activity. Interestingly, conditional overexpression of PIASy selectively inhibits a subset of endogenous TGF-β-responsive genes, which includes the cyclin-dependent kinase inhibitor p15, and the plasminogen activator inhibitor 1. We further show that PIASy can interact constitutively with histone deacetylase 1 (HDAC1) and that addition of HDAC inhibitor trichostatin A (TSA) can prevent the inhibitory function of PIASy. Taken together, our studies indicate that PIASy can inhibit TGF-β/Smad transcriptional responses through interactions with Smad proteins and HDAC.

Activation of Smad transcriptional activity by protein inhibitor of activated STAT3 (PIAS3).

Smad proteins play pivotal roles in mediating the transforming growth factor β (TGF-β) transcriptional responses. We show in this report that PIAS3, a member of the protein inhibitor of activated STAT (PIAS) family, activates TGF-β/Smad transcriptional responses. PIAS3 interacts with Smad proteins, most strongly with Smad3. PIAS3 and Smad3 interact with each other at the endogenous protein level in mammalian cells and also in vitro, and the association occurs through the C-terminal domain of Smad3. We further show that PIAS3 can interact with the general coactivators p300/CBP, the first evidence that a PIAS protein can associate with p300/CBP. In contrast, PIASy, which inhibits Smad transcriptional activity and other transcriptional responses, is unable to interact with p300/CBP. The RING domain of PIAS3 is essential for interaction with p300/CBP, and a RING domain mutant PIAS3, which cannot bind p300/CBP, no longer activates TGF-β/Smad-dependent transcription. Furthermore, we show that PIAS3, Smad3, and p300 can form a ternary complex, which is markedly increased by TGF-β treatment. Taken together, our studies indicate that on TGF-β treatment, PIAS3 can form a complex with Smads and p300/CBP and activate Smad transcriptional activity.

Transforming Growth Factor-β-inducible Phosphorylation of Smad3

Smad proteins transduce the transforming growth factor-β (TGF-β) signal at the cell surface into gene regulation in the nucleus. Upon TGF-β treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-β receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser208, Ser204, and Thr179 are phosphorylated in response to TGF-β. The linker phosphorylation peaks at 1 h after TGF-β treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-β receptor is necessary for the TGF-β-induced linker phosphorylation. Although the TGF-β receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-β-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-β-inducible Ser204 phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-β-induced phosphorylation of Thr179 and Ser208, suggesting that the CDK family is responsible for phosphorylation of Thr179 and Ser208 in response to TGF-β. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-β/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-β-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity.

Pin1 Promotes Transforming Growth Factor-β-induced Migration and Invasion

Transforming growth factor-β (TGF-β) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-β responses. TGF-β binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-β-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-β, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-β-induced growth-inhibitory responses and a number of TGF-β/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-β-mediated migration and invasion. Accordingly, TGF-β induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-β-induced migration and invasion of cancer cells.

The Smad3 linker region contains a transcriptional activation domain

Transforming growth factor-beta (TGF-beta)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-beta/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-beta transcriptional activation responses, although it can be phosphorylated by the TGF-beta receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-beta. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control.

Functional interaction between Smad3 and S100A4 (metastatin-1) for TGF-β-mediated cancer cell invasiveness

TGF-beta (transforming growth factor-beta) induces a cytostatic response in most normal cell types. In cancer cells, however, it often promotes metastasis, and its high expression is correlated with poor prognosis. In the present study, we show that S100A4, a metastasis-associated protein, also called metastatin-1, can physically and functionally interact with Smad3, an important mediator of TGF-beta signalling. In agreement with its known property, S100A4 binds to Smad3 in a Ca2+-dependent manner. The S100A4-binding site is located in the N-terminal region of Smad3. S100A4 can potentiate transcriptional activity of Smad3 and the related Smad2. When exogenously expressed in MCF10CA1a.cl1, an MCF10-derived breast cancer cell line, S100A4 increases TGF-beta-induced MMP-9 (matrix metalloproteinase-9) expression. On the other hand, depletion of S100A4 by siRNA (small interfering RNA) from the MDA-MB231 cell line results in attenuation of MMP-9 induction by TGF-beta. Consistent with these observations, S100A4 increases cell invasion ability induced by TGF-beta in MCF10CA1a.cl1 cells, and depletion of the protein in MDA-MB-231 cells inhibits it. Because expression of both S100A4 and TGF-beta is highly elevated in many types of malignant tumours, S100A4 and Smad3 may co-operatively increase metastatic activity of some types of cancer cells.

Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase I. IDENTIFICATION OF THE KINASE AND ITS ROLE IN THE TURNOFF OF PHOSPHODIESTERASE IN VITRO

Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by Pγ, the regulatory subunit of PDE, and GTP/Tα, the GTP-bound α subunit of transducin. In previous studies (Tsuboi, S., Matsumoto, H., Jackson, K. W., Tsujimoto, K., Williamas, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016–15023; Tsuboi, S., Matsumoto, H., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15024–15029), we showed that Pγ is phosphorylated by a previously unknown kinase (Pγ kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated Pγ loses its ability to interact with GTP/Tα, but gains a 10–15 times higher ability to inhibit GTP/Tα-activated PDE than that of nonphosphorylated Pγ. Thus, we propose that the Pγ phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/Tα-activated PDE. Here we demonstrate that all known Pγs preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that Pγ kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of Pγ indicates that all known Pγs contain a P-X-T-P-R sequence and that this sequence is required for the Pγ phosphorylation by Pγ kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the Pγ kinase activity exactly coelutes with Cdk5 and p35. The Pγ kinase activity (∼85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates Pγ. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates Pγ in frog outer segment membranes in a GTP-dependent manner. These observations suggest that Cdk5 is probably involved in the recovery phase of phototransduction through phosphorylation of Pγ complexed with GTP/Tα in mature vertebrate retinal photoreceptors.

Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase II. ITS ROLE IN THE TURNOFF OF PHOSPHODIESTERASE IN VIVO

Retinal cGMP phosphodiesterase (PDE) is regulated by Pγ, the regulatory subunit of PDE, and GTP/Tα, the GTP-bound α subunit of transducin. In the accompanying paper (Matsuura, I., Bondarenko, V. A., Maeda, T., Kachi, S., Yamazaki, M., Usukura, J., Hayashi, F., and Yamazaki, A. (2000) J. Biol. Chem. 275, 32950–32957), we have shown that all known Pγs contain a specific phosphorylation motif for cyclin-dependent protein kinase 5 (Cdk5) and that the unknown kinase is Cdk5 complexed with its activator. Here, using frog rod photoreceptor outer segments (ROS) isolated by a new method, we show that Cdk5 is involved in light-dependent Pγ phosphorylation in vivo. Under dark conditions only negligible amounts of Pγ were phosphorylated. However, under illumination that bleached less than 0.3% of the rhodopsin, ∼4% of the total Pγ was phosphorylated in less than 10 s. Pγ dephosphorylation occurred in less than 1 s after the light was turned off. Analysis of the phosphorylated amino acid, inhibition of Pγ phosphorylation by Cdk inhibitors in vivo and in vitro, and two-dimensional peptide map analysis of Pγ phosphorylated in vivo and in vitro indicate that Cdk5 phosphorylates a Pγ threonine in the same manner in vivo and in vitro. These observations, together with immunological data showing the presence of Cdk5 in ROS, suggest that Cdk5 is involved in light-dependent Pγ phosphorylation in ROS and that the phosphorylation is significant and reversible. In an homogenate of frog ROS, PDE activated by light/guanosine 5′-O-(3-thiotriphosphate) (GTPγS) was inhibited by Pγ alone, but not by Pγ complexed with GDP/Tα or GTPγS/Tα. Under these conditions, Pγ phosphorylated by Cdk5 inhibited the light/GTPγS-activated PDE even in the presence of GTPγS/Tα. These observations suggest that phosphorylated Pγ interacts with and inhibits light/GTPγS-activated PDE, but does not interact with GTPγS/Tα in the homogenate. Together, our results strongly suggest that after activation of PDE by light/GTP, Pγ is phosphorylated by Cdk5 and the phosphorylated Pγ inhibits GTP/Tα-activated PDE, even in the presence of GTP/Tα in ROS.

Novel IL31RA gene mutation and ancestral OSMR mutant allele in familial primary cutaneous amyloidosis

Primary cutaneous amyloidosis (PCA) is an itchy skin disorder associated with amyloid deposits in the superficial dermis. The disease is relatively common in Southeast Asia and South America. Autosomal dominant PCA has been mapped earlier to 5p13.1–q11.2 and two pathogenic missense mutations in the OSMR gene, which encodes the interleukin-6 family cytokine receptor oncostatin M receptor beta (OSMRβ), were reported. Here, we investigated 29 Taiwanese pedigrees with PCA and found that 10 had heterozygous missense mutations in OSMR: p.D647V (one family), p.P694L (six families), and p.K697T (three families). The mutation p.P694L was associated with the same haplotype in five of six families and also detected in two sporadic cases of PCA. Of the other 19 pedigrees that lacked OSMR pathology, 8 mapped to the same locus on chromosome 5, which also contains the genes for 3 other interleukin-6 family cytokine receptors, including interleukin-31 receptor A (IL31RA), which can form a heterodimeric receptor with OSMRβ through interleukin-31 signaling. In one family, we identified a point mutation in the IL31RA gene, c.1562C>T that results in a missense mutation, p.S521F, which is also sited within a fibronectin type III-like repeat domain as observed in the OSMR mutations. PCA is a genetically heterogeneous disorder but our study shows that it can be caused by mutations in two biologically associated cytokine receptor genes located on chromosome 5. The identification of OSMR and IL31RA gene pathology provides an explanation of the high prevalence of PCA in Taiwan as well as new insight into disease pathophysiology.