Amelogenin phosphorylation regulates tooth enamel formation by stabilizing a transient amorphous mineral precursor.

Dental enamel comprises interwoven arrays of extremely long and narrow crystals of carbonated hydroxyapatite called enamel rods. Amelogenin (AMELX) is the predominant extracellular enamel matrix protein and plays an essential role in enamel formation (amelogenesis). Previously, we have demonstrated that full-length AMELX forms higher-order supramolecular assemblies that regulate ordered mineralization in vitro, as observed in enamel rods. Phosphorylation of the sole AMELX phosphorylation site (Ser-16) in vitro greatly enhances its capacity to stabilize amorphous calcium phosphate (ACP), the first mineral phase formed in developing enamel, and prevents apatitic crystal formation. To test our hypothesis that AMELX phosphorylation is critical for amelogenesis, we generated and characterized a hemizygous knockin (KI) mouse model with a phosphorylation-defective Ser-16 to Ala-16 substitution in AMELX. Using EM analysis, we demonstrate that in the absence of phosphorylated AMELX, KI enamel lacks enamel rods, the hallmark component of mammalian enamel, and, unlike WT enamel, appears to be composed of less organized arrays of shorter crystals oriented normal to the dentinoenamel junction. KI enamel also exhibited hypoplasia and numerous surface defects, whereas heterozygous enamel displayed highly variable mosaic structures with both KI and WT features. Importantly, ACP-to-apatitic crystal transformation occurred significantly faster in KI enamel. Secretory KI ameloblasts also lacked Tomes' processes, consistent with the absence of enamel rods, and underwent progressive cell pathology throughout enamel development. In conclusion, AMELX phosphorylation plays critical mechanistic roles in regulating ACP-phase transformation and enamel crystal growth, and in maintaining ameloblast integrity and function during amelogenesis.

PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization.

We have shown a novel mechanism of Akt-mediated regulation of the CDK inhibitor p27(kip1). Blockade of HER2/neu in tumor cells inhibits Akt kinase activity and upregulates nuclear levels of the CDK inhibitor (Kip1). Recombinant Akt and Akt precipitated from tumor cells phosphorylated wild-type p27 in vitro. p27 contains an Akt consensus RXRXXT(157)D within its nuclear localization motif. Active (myristoylated) Akt phosphorylated wild-type p27 in vivo but was unable to phosphorylate a T157A-p27 mutant. Wild-type p27 localized in the cytosol and nucleus, whereas T157A-p27 localized exclusively in the nucleus and was resistant to nuclear exclusion by Akt. T157A-p27 was more effective than wild-type p27 in inhibiting cyclin E/CDK2 activity and cell proliferation; these effects were not rescued by active Akt. Expression of Ser(473) phospho Akt in primary human breast cancers statistically correlated with expression of p27 in tumor cytosol. These data indicate that Akt may contribute to tumor-cell proliferation by phosphorylation and cytosolic retention of p27, thus relieving CDK2 from p27-induced inhibition.

Role of RUNX Family Transcription Factors in DNA Damage Response.

Cells are constantly exposed to endogenous and exogenous stresses that can result in DNA damage. In response, they have evolved complex pathways to maintain genomic integrity. RUNX family transcription factors (RUNX1, RUNX2, and RUNX3 in mammals) are master regulators of development and differentiation, and are frequently dysregulated in cancer. A growing body of research also implicates RUNX proteins as regulators of the DNA damage response, often acting in conjunction with the p53 and Fanconi anemia pathways. In this review, we discuss the functional role and mechanisms involved in RUNX factor mediated response to DNA damage and other cellular stresses. We highlight the impact of these new findings on our understanding of cancer predisposition associated with RUNX factor dysregulation and their implications for designing novel approaches to prevent cancer formation in affected individuals.

p130CAS is required for netrin signaling and commissural axon guidance.

Netrins are an important family of axon guidance cues. Here, we report that netrin-1 induces tyrosine phosphorylation of p130(CAS) (Crk-associated substrate). Our biochemical studies indicate that p130(CAS) is downstream of the Src family kinases and upstream of the small GTPase Rac1 and Cdc42. Inhibition of p130(CAS) signaling blocks both the neurite outgrowth-promoting activity and the axon attraction activity of netrin-1. p130(CAS) RNA interference inhibits the attraction of commissural axons in the spinal cord by netrin-1 and causes defects in commissural axon projection in the embryo. These results demonstrate that p130(CAS) is a key component in the netrin signal transduction pathway and plays an important role in guiding commissural axons in vivo.

Crk-associated substrate tyrosine phosphorylation sites are critical for invasion and metastasis of SRC-transformed cells.

Crk-associated substrate (CAS, p130Cas) is a major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. We recently reported that reexpression of CAS in CAS-deficient mouse embryo fibroblasts transformed by oncogenic Src promoted an invasive phenotype associated with enhanced cell migration through Matrigel, organization of actin into large podosome ring and belt structures, activation of matrix metalloproteinase-2, and elevated tyrosine phosphorylation of the focal adhesion proteins FAK and paxillin. We have now extended these studies to examine the mechanism by which CAS achieves these changes and to evaluate the potential role for CAS in promoting in vivo tumor growth and metastasis. Whereas the presence or absence of CAS did not alter the primary growth of subcutaneous-injected Src-transformed mouse embryo fibroblasts, CAS expression was required to promote lung metastasis following removal of the primary tumor. The substrate domain YxxP tyrosines, the major sites of CAS phosphorylation by Src that mediate interactions with Crk, were found to be critical for promoting both invasive and metastatic properties of the cells. The ability of CAS to promote Matrigel invasion, formation of large podosome structures, and tyrosine phosphorylation of Src substrates, including FAK, paxillin, and cortactin, was also strictly dependent on the YxxP tyrosines. In contrast, matrix metalloproteinase-2 activation was most dependent on the CAS SH3 domain, whereas the substrate domain YxxP sites also contributed to this property. Thus multiple CAS-mediated signaling events are implicated in promoting invasive and metastatic properties of Src-transformed cells.

The Actin-Binding Protein Cofilin and Its Interaction With Cortactin Are Required for Podosome Patterning in Osteoclasts and Bone Resorption In Vivo and In Vitro.

The adhesion of osteoclasts (OCs) to bone and bone resorption require the assembly of specific F-actin adhesion structures, the podosomes, and their dense packing into a sealing zone. The OC-specific formation of the sealing zone requires the interaction of microtubule (MT) + ends with podosomes. Here, we deleted cofilin, a cortactin (CTTN)- and actin-binding protein highly expressed in OCs, to determine if it acts downstream of the MT-CTTN axis to regulate actin polymerization in podosomes. Conditional deletion of cofilin in OCs in mice, driven by the cathepsin K promoter (Ctsk-Cre), impaired bone resorption in vivo, increasing bone density. In vitro, OCs were not able to organize podosomes into peripheral belts. The MT network was disorganized, MT stability was decreased, and cell migration impaired. Active cofilin stabilizes MTs and allows podosome belt formation, whereas MT disruption deactivates cofilin via phosphorylation. Cofilin interacts with CTTN in podosomes and phosphorylation of either protein disrupts this interaction, which is critical for belt stabilization and for the maintenance of MT dynamic instability. Accordingly, active cofilin was required to rescue the OC cytoskeletal phenotype in vitro. These findings suggest that the patterning of podosomes into a sealing zone involves the dynamic interaction between cofilin, CTTN, and the MTs + ends. This interaction is critical for the functional organization of OCs and for bone resorption. © 2016 American Society for Bone and Mineral Research.

CAS promotes invasiveness of Src-transformed cells.

CAS ('Crk-associated substrate') is an Src substrate found at sites of integrin-mediated cell adhesion and linked to cell motility and survival. In this study, the involvement of CAS in oncogenic transformation was evaluated through analysis of mouse embryo fibroblast populations expressing an activated Src mutant, either in the presence or absence of CAS expression. CAS was not found to be a critical determinant of either Src-mediated morphologic transformation or anchorage-independent growth. However, CAS had a profound effect on other aspects of oncogenic Src function. CAS expression led to a substantial increase in the phosphotyrosine content of FAK and paxillin, supporting a role for CAS as a positive regulator of Src activity at integrin adhesion sites. Importantly, CAS expression resulted in a striking enhancement of the capacity of Src-transformed cells to invade through Matrigel. The increased invasiveness was associated with increased activation of matrix metalloproteinase MMP-2 and formation of large actin-rich podosomal aggregates appearing as ring and belt structures. Thus, elevated CAS-associated tyrosine phosphorylation signaling events occurring at sites of integrin-mediated cell adhesion can have a major role in the development of an invasive cell phenotype.

Regulation and localization of CAS substrate domain tyrosine phosphorylation.

Crk-associated substrate (CAS) is a tyrosine kinase substrate implicated in integrin control of cell behavior. Phosphorylation, by Src family kinases, of multiple tyrosine residues in the CAS substrate domain (SD) is a major integrin signaling event that promotes cell motility. In this study, novel phosphospecific antibodies directed against CAS SD phosphotyrosine sites ("pCAS" antibodies) were characterized and employed to investigate the cellular regulation and localization of CAS SD tyrosine phosphorylation. An analysis of CAS and focal adhesion kinase (FAK) variants expressed in CAS- and FAK-deficient cell lines, respectively, indicated that CAS SD tyrosine phosphorylation is substantially achieved by Src family kinases brought into association with CAS through two distinct mechanisms: direct binding to the CAS Src-binding domain and indirect association through a FAK bridge. Cell immunostaining with pCAS antibodies revealed that CAS SD tyrosine phosphorylation occurs exclusively at sites of integrin adhesion including both nascent focal complexes formed at the edges of extending lamellipodia as well as mature focal adhesions underlying the cell body. These findings further document a role for FAK as an important upstream regulator of CAS SD tyrosine phosphorylation and implicate CAS-mediated signaling events in promoting membrane protrusion/lamellipodium extension during cell motility.

Focal adhesion kinase signaling activities and their implications in the control of cell survival and motility.

Focal adhesion kinase (FAK) was first described in 1992 as a novel nonreceptor protein-tyrosine kinase localized prominently within focal adhesions, suggesting a signaling role in regulating cell behavior resulting from integrin interaction with the extracellular matrix. Subsequent studies over the past decade have established functional roles for FAK as a positive regulator of both cell motility and cell survival, while providing considerable insight into signaling mechanisms involved. FAK signaling results from its ability to become highly phosphorylated in response to integrin-mediated adhesion on Tyr-397, permitting interactions with a number of different signaling effectors containing Src homology 2 (SH2) domains. Src-family kinases recruited to the Tyr-397 site phosphorylate two FAK-interacting proteins, Crk-associated substrate (CAS) and paxillin, which results ultimately in regulation of Rho-family GTPases contributing to cell motility. CAS phosphorylation, as well as phosphatidylinositol 3-kinase (PI3K) activation resulting from its binding to the FAK Tyr-397 site, have been implicated as downstream FAK signaling events that confer a resistance to apoptosis. This article reviews these and other aspects of FAK signaling and function.

Dynamin and endocytosis are required for the fusion of osteoclasts and myoblasts.

Cell-cell fusion is an evolutionarily conserved process that leads to the formation of multinucleated myofibers, syncytiotrophoblasts and osteoclasts, allowing their respective functions. Although cell-cell fusion requires the presence of fusogenic membrane proteins and actin-dependent cytoskeletal reorganization, the precise machinery allowing cells to fuse is still poorly understood. Using an inducible knockout mouse model to generate dynamin 1- and 2-deficient primary osteoclast precursors and myoblasts, we found that fusion of both cell types requires dynamin. Osteoclast and myoblast cell-cell fusion involves the formation of actin-rich protrusions closely associated with clathrin-mediated endocytosis in the apposed cell. Furthermore, impairing endocytosis independently of dynamin also prevented cell-cell fusion. Since dynamin is involved in both the formation of actin-rich structures and in endocytosis, our results indicate that dynamin function is central to the osteoclast precursors and myoblasts fusion process, and point to an important role of endocytosis in cell-cell fusion.

The integrin-coupled signaling adaptor p130Cas suppresses Smad3 function in transforming growth factor-beta signaling.

Reciprocal cooperative signaling by integrins and growth factor receptors at G1 phase during cell cycle progression is well documented. By contrast, little is known about the cross-talk between integrin and transforming growth factor (TGF)-beta signaling. Here, we show that integrin signaling counteracts the inhibitory effects of TGF-beta on cell growth and that this effect is mediated by p130Cas (Crk-associated substrate, 130 kDa). Adhesion to fibronectin or laminin reduces TGF-beta-induced Smad3 phosphorylation and thus inhibits TGF-beta-mediated growth arrest; loss of p130Cas abrogates these effects. Loss and gain of function studies demonstrated that, once tyrosine-phosphorylated via integrin signaling, p130Cas binds to Smad3 and reduces phosphorylation of Smad3. That in turn leads to inhibition of p15 and p21 expression and facilitation of cell cycle progression. Thus, p130Cas-mediated control of TGF-beta/Smad signaling may provide an additional clue to the mechanism underlying resistance to TGF-beta-induced growth inhibition.

Protein targets of reactive electrophiles in human liver microsomes.

Liver microsomes are widely used to study xenobiotic metabolism in vitro, and covalent binding to microsomal proteins serves as a surrogate marker for toxicity mediated by reactive metabolites. We have applied liquid chromatography-tandem mass spectrometry (LC-MS-MS) to identify protein targets of the biotin-tagged model electrophiles 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) in human liver microsomes. The biotin-tagged peptides resulting from in-gel tryptic digestion were enriched by biotin-avidin chromatography and LC-MS-MS was used to identify 376 microsomal cysteine thiol targets of BMCC and IAB in 263 proteins. Protein adduction was selective and reproducible, and only 90 specific cysteine sites in 70 proteins (approximately 25% of the total) were adducted by both electrophiles. Differences in adduction selectivity correlated with different biological effects of the compounds, as IAB- but not BMCC-induced ER stress in HEK293 cells. Targeted LC-MS-MS analysis of microsomal glutathione-S-transferase cysteine 50, a target of both IAB and BMCC, detected time-dependent adduction by the reactive acetaminophen metabolite N-acetyl-p-benzoquinoneimine during microsomal incubations. The results indicate that electrophiles selectively adduct microsomal proteins, but display differing target selectivities that correlate with differences in toxicity. Analysis of selected microsomal protein adduction reactions thus could provide a more specific indication of potential toxicity than bulk covalent binding of radiolabeled compounds.

Subsets of the major tyrosine phosphorylation sites in Crk-associated substrate (CAS) are sufficient to promote cell migration.

Crk-associated substrate (p130(CAS) or CAS) is a major integrin-associated Src substrate that undergoes tyrosine phosphorylation at multiple YXXP motifs in its substrate domain (SD) to create docking sites for SH2-containing signaling effectors. Notably, recruitment of Crk adaptor proteins to the CAS SD sites is implicated in promoting cell migration. However, it is unclear which or how many of the 15 CAS SD YXXP tyrosines are critically involved. To gain a better understanding of CAS SD function, we assessed the signaling capacity of individual YXXP motifs. Using site-directed mutagenesis combined with tryptic phosphopeptide mapping, we determined that the ten tyrosines in YXXP motifs 6-15 are the major sites of CAS SD phosphorylation by Src. Phosphopeptide binding assays showed that all of these sites are capable of binding the Crk SH2 domain. To evaluate the requirement for CAS YXXP sites in stimulating cell migration, a series of phenylalanine substitution variants were expressed in CAS -/- mouse embryo fibroblasts. CAS expression enhanced the rate of cell migration into a monolayer wound in a manner dependent on the major sites of Src phosphorylation. Effective wound healing was achieved by CAS variants containing as few as four of the major sites, indicating sufficiency of partial SD signaling function in this cell migration response.