Marcelo Antonelli

A noncanonical sequence phosphorylated by casein kinase 1 in beta-catenin may play a role in casein kinase 1 targeting of important signaling proteins.

Protein kinase casein kinase 1 (CK1) phosphorylates Ser-45 of β-catenin, “priming” the subsequent phosphorylation by glycogen synthase-3 of residues 41, 37, and 33. This concerted phosphorylation of β-catenin signals its degradation and prevents its function in triggering cell division. The sequence around Ser-45 does not conform to the canonical consensus for CK1 substrates, which prescribes either phosphoamino acids or acidic residues in position n-3 from the target serine. However, the β-catenin sequence downstream from Ser-45 is very similar to a sequence recognized by CK1 in nuclear factor for activated T cells 4. The common features include an SLS motif followed two to five residues downstream by a cluster of acidic residues. Synthetic peptides reproducing residues 38-65 of β-catenin were assayed with purified rat liver CK1 or recombinant CK1α and CK1αL from zebrafish. The results demonstrate that SLS and acidic cluster motifs are crucial for CK1 recognition. Pro-44 and Pro-52 are also important for efficient phosphorylation. Similar results were obtained with the different isoforms of CK1. Phosphorylation of mutants of full-length recombinant β-catenin from zebrafish confirmed the importance of the SLS and acidic cluster motifs. A search for proteins with similar motifs yielded, among other proteins, adenomatous polyposis coli, previously found to be phosphorylated by CK1. There is a strong correlation of β-catenin mutations found in thyroid tumors with the motifs recognized by CK1 in this protein.

The cancer-related transcription factor Runx2 modulates cell proliferation in human osteosarcoma cell lines.

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre‐osteoblast proliferation by affecting cell cycle progression in the G1 phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G1/S phase transition, and Runx2 is again up‐regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle‐regulated with respect to the G1/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre‐established levels in a given cell type triggers one or more anti‐proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. J. Cell. Physiol. 228: 714–723, 2013.

Phosphorylation of AKT/PKB by CK2 is necessary for the AKT-dependent up-regulation of β-catenin transcriptional activity

β‐Catenin is a key protein in the canonical Wnt signaling pathway and in many cancers alterations in transcriptional activity of its components are observed. This pathway is up‐regulated by the protein kinase CK2, but the underlying mechanism of this change is unknown. It has been demonstrated that CK2 hyperactivates AKT/PKB by phosphorylation at Ser129, and AKT phosphorylates β‐catenin at Ser552, which in turn, promotes its nuclear localization and transcriptional activity. However, the consequences of CK2‐dependent hyperactivation of AKT on β‐catenin activity and cell viability have not been evaluated. We assessed this regulatory process by manipulating the activity of CK2 and AKT through overexpression of wild‐type, constitutively active and dominant negative forms of these proteins as well as analyzing β‐catenin‐dependent transcriptional activity, survivin expression and viability in HEK‐293T cells. We observed that CK2α overexpression up‐regulated the β‐catenin transcriptional activity, which correlated to an increased nuclear localization of β‐catenin as well as survivin expression. Importantly, these effects were strongly reversed when an AKT‐S129A mutant was co‐expressed in the same cells, followed by a significant decrease in cell viability but no changes in β‐catenin stability. Taken together, the data suggest that the CK2α‐dependent up‐regulation of β‐catenin activity requires phosphorylation of AKT in human embryonic kidney cells. J. Cell. Physiol. 226: 1953–1959, 2011.

Impaired Cell Cycle Regulation of the Osteoblast-Related Heterodimeric Transcription Factor Runx2-Cbfβ in Osteosarcoma Cells

Bone formation and osteoblast differentiation require the functional expression of the Runx2/Cbfβ heterodimeric transcription factor complex. Runx2 is also a suppressor of proliferation in osteoblasts by attenuating cell cycle progression in G1. Runx2 levels are modulated during the cell cycle, which are maximal in G1 and minimal beyond the G1/S phase transition (S, G2, and M phases). It is not known whether Cbfβ gene expression is cell cycle controlled in preosteoblasts nor how Runx2 or Cbfβ are regulated during the cell cycle in bone cancer cells. We investigated Runx2 and Cbfβ gene expression during cell cycle progression in MC3T3‐E1 osteoblasts, as well as ROS17/2.8 and SaOS‐2 osteosarcoma cells. Runx2 protein levels are reduced as expected in MC3T3‐E1 cells arrested in late G1 (by mimosine) or M phase (by nocodazole), but not in cell cycle arrested osteosarcoma cells. Cbfβ protein levels are cell cycle independent in both osteoblasts and osteosarcoma cells. In synchronized MC3T3‐E1 osteoblasts progressing from late G1 or mitosis, Runx2 levels but not Cbfβ levels are cell cycle regulated. However, both factors are constitutively elevated throughout the cell cycle in osteosarcoma cells. Proteasome inhibition by MG132 stabilizes Runx2 protein levels in late G1 and S in MC3T3‐E1 cells, but not in ROS17/2.8 and SaOS‐2 osteosarcoma cells. Thus, proteasomal degradation of Runx2 is deregulated in osteosarcoma cells. We propose that cell cycle control of Runx2 gene expression is impaired in osteosarcomas and that this deregulation may contribute to the pathogenesis of osteosarcoma. J. Cell. Physiol. 221: 560–571, 2009.

Biochemical and cellular characteristics of the four splice variants of protein kinase CK1α from zebrafish (Danio rerio)

Protein kinase CK1 (previously known as casein kinase I) conforms to a subgroup of the great protein kinase family found in eukaryotic organisms. The CK1 subgroup of vertebrates contains seven members known as α, β, γ1, γ2, γ3, δ, and ε. The CK1α gene can generate four variants (CK1α, CK1αS, CK1αL, and CK1αLS) through alternate splicing, characterized by the presence or absence of two additional coding sequences. Exon “L” encodes a 28‐amino acid stretch that is inserted after lysine 152, in the center of the catalytic domain. The “S” insert encodes 12 amino acid residues and is located close to the carboxyl terminus of the protein. This work reports some biochemical and cellular properties of the four CK1α variants found to be expressed in zebrafish (Danio rerio). The results obtained indicate that the presence of the “L” insert affects several biochemical properties of CK1α: (a) it increases the apparent Km for ATP twofold, from ∼30 to ∼60 μM; (b) it decreases the sensitivity to the CKI‐7 inhibitor, raising the I50 values from 113 to ∼230 μM; (c) it greatly decreases the heat stability of the enzyme at 40°C. In addition, the insertion of the “L” fragment exerts very important effects on some cellular properties of the enzyme. CK1αL concentrates in the cell nucleus, excluding nucleoli, while the CK1α variant is predominantly cytoplasmic, although some presence is observed in the nucleus. This finding supports the thesis that the basic‐rich region found in the “L” insert acts as a nuclear localization signal. The “L” insert‐containing variant was also found to be more rapidly degraded (half‐life of 100 min) than the CK1α variant (half‐life of 400 min) in transfected Cos‐7 cells. J. Cell. Biochem. 86: 805–814, 2002.

CK2 functionally interacts with AKT/PKB to promote the β-catenin-dependent expression of survivin and enhance cell survival

Abstractβ-Catenin is crucial in the canonical Wnt signaling pathway. This pathway is up-regulated by CK2 which is associated with an enhanced expression of the antiapoptotic protein survivin, although the underlying molecular mechanism is unknown. AKT/PKB kinase phosphorylates and promotes β-catenin transcriptional activity, whereas CK2 hyperactivates AKT by phosphorylation at Ser129; however, the role of this phosphorylation on β-catenin transcriptional activity and cell survival is unclear. We studied in HEK-293T cells, the effect of CK2-dependent hyperactivation of AKT on cell viability, as well as analyzed β-catenin subcellular localization and transcriptional activity and survivin expression. CK2α overexpression led to an augmented β-catenin-dependent transcription and protein levels of survivin, and consequently an enhanced resistance to apoptosis. However, CK2α-enhancing effects were reversed when an AKT mutant deficient in Ser129 phosphorylation by CK2 was co-expressed. Therefore, our results strongly suggest that CK2α-specific enhancement of β-catenin transcriptional activity as well as cell survival may depend on AKT hyperactivation by CK2.

Human-Xenopus chimeras of Gsα reveal a new region important for its activation of adenylyl cyclase

G proteins are heterotrimeric GTPases that play a key role in signal transduction. The α subunit of Gs bound to GTP is capable of activating adenylyl cyclase. The amino acid sequences derived from two X. laevis cDNA clones that apparently code for Gsα subunits are 92% identical to those found in the short form of human Gsα. Despite this high homology, the X. laevis Gsα clones expressed in vitro, yielded a protein that are not able to activate the adenylyl cyclase present in S49 cyc− membranes in contrast with human Gsα similarly expressed. This finding suggested that the few amino acid substitutions found in the amphibian subunit are important in defining the functionality of the human Gsα. The construction of chimeras composed of different fractions of the cDNAs of the two species was adopted as an approach in determining the regions of the molecule important in its functionality in this assay. Four pairs of chimeras were constructed using reciprocal combinations of the cDNAs coding for human and Xenopus Gsα. These eight constructs were expressed in vitro and equivalent amounts of the resulting proteins were assayed in the activation of adenylyl cyclase with GTPγs and isoproterenol. The results obtained here clearly indicate that the Gα sequence that extends from amino acid 70 to 140, is important for the functionality of human Gsα in activating adenylyl cyclase.

An inactive mutant of the α subunit of protein kinase CK2 that traps the regulatory CK2β subunit

Protein kinase CK2 (casein kinase 2) is a ubiquitous Ser/Thr protein kinase involved in cell proliferation. Mutation of the α subunit of the Xenopus laevis CK2 to change aspartic acid 156 to alanine (CK2αA156) resulted in an inactive enzyme. The CK2αA156 mutant, however, binds the regulatory subunit as measured by retention of β on a nickel chelating column mediated by (His)6‐tagged CK2αA156. Addition of CK2αA156 also caused β to shift sedimentation in a sucrose gradient from a β 2 dimer (52 kDa) to an α 2 β 2 tetramer (130 000 kDa). CK2αA156 can trap the β subunit in an inactive complex reducing the stimulation of casein phosphorylation caused by addition of β to wild‐type α. This competitive effect depends on the ratio of α/αA156 and on the amount of β available. Since β inhibits the phosphorylation of calmodulin by CK2α, the addition of CK2αA156, in this case, increases calmodulin phosphorylation by the α and β combination. These results suggest that CK2αA156 may be a useful dominant‐negative mutant that can serve to explore the multiple functions of CK2β.

TACE/ADAM17 is involved in germ cell apoptosis during rat spermatogenesis

The pathways leading to male germ cell apoptosis in vivo are poorly understood, but are highly relevant for the comprehension of sperm production regulation by the testis. In this work, we show the evidence of a mechanism where germ cell apoptosis is induced through the inactivation and shedding of the extracellular domain of KIT (c-kit) by the protease TACE/a disintegrin and metalloprotease 17 (ADAM17) during the first wave of spermatogenesis in the rat. We show that germ cells undergoing apoptosis lacked the extracellular domain of the KIT receptor. TACE/ADAM17, a membrane-bound metalloprotease, was highly expressed in germ cells undergoing apoptosis as well. On the contrary, cell surface presence of ADAM10, a closely related metalloprotease isoform, was not associated with apoptotic germ cells. Pharmacological inhibition of TACE/ADAM17, but not ADAM10, significantly prevented germ cell apoptosis in the male pubertal rat. Induction of TACE/ADAM17 by the phorbol-ester phorbol 12-myristate 13-acetate (PMA) induced germ cell apoptosis, which was prevented when an inhibitor of TACE/ADAM17 was present in the assay. Ex-vivo rat testis culture showed that PMA induced the cleavage of the KIT extracellular domain. Isolation of apoptotic germ cells showed that even though protein levels of TACE/ADAM17 were higher in apoptotic germ cells than in nonapoptotic cells, the contrary was observed for ADAM10. These results suggest that TACE/ADAM17 is one of the elements triggering physiological germ cell apoptosis during the first wave of spermatogenesis.

Xenopus laevis oocyte Gα subunits mRNAs Detection and quantitation during oogenesis and early embryogenesis by competitive reverse PCR

The expression of mRNAs coding for different Xenopus laevis oocyte Gα subunits was analyzed by the PCR technique. Using the nucleotide sequences of five previously cloned cDNAs for oocyte Gα subunits [FEBS Lett. 244, 188–192, 1989; FEBS Lett. 268, 27–31, 1990] and the highly sensitive reverse PCR reaction we found that Gαo, Gαi‐1, Gαi‐3 and Gαs species are present in oocyte stage VI, Gαo mRNA being the most abundant transcript. Gαo mRNA was further quantitated through oogenesis, unfertilized eggs and early embryogenesis stages by a competitive PCR reaction using an ‘in vitro’ deleted Gαo mRNA as the internal standard. Using this approach we found that Xenopus Gαo mRNA levels were constant during oogenesis and unfertilized eggs at a concentration of 3.5 pg of mRNA/stage (5 × 105 molecules) and diminish gradually during early embryogenesis, reaching a level of 0.3 pg in the gastrula stage. These findings show that oocyte Gαo, and perhaps the rest of the α subunits, are expressed as maternal mRNAs and could play an important role in signal transduction at the beginning of oocyte cell differentiation.