Tyrosine phosphorylation of type Iγ phosphatidylinositol phosphate kinase by Src regulates an integrin–talin switch

Engagement of integrin receptors with the extracellular matrix induces the formation of focal adhesions (FAs). Dynamic regulation of FAs is necessary for cells to polarize and migrate. Key interactions between FA scaffolding and signaling proteins are dependent on tyrosine phosphorylation. However, the precise role of tyrosine phosphorylation in FA development and maturation is poorly defined. Here, we show that phosphorylation of type Iγ phosphatidylinositol phosphate kinase (PIPKIγ661) on tyrosine 644 (Y644) is critical for its interaction with talin, and consequently, localization to FAs. PIPKIγ661 is specifically phosphorylated on Y644 by Src. Phosphorylation is regulated by focal adhesion kinase, which enhances the association between PIPKIγ661 and Src. The phosphorylation of Y644 results in an ∼15-fold increase in binding affinity to the talin head domain and blocks β-integrin binding to talin. This defines a novel phosphotyrosine-binding site on the talin F3 domain and a “molecular switch” for talin binding between PIPKIγ661 and β-integrin that may regulate dynamic FA turnover

Dominant Role of Oncogene Dosage and Absence of Tumor Suppressor Activity in Nras-Driven Hematopoietic Transformation

Biochemical properties of Ras oncoproteins and their transforming ability strongly support a dominant mechanism of action in tumorigenesis. However, genetic studies unexpectedly suggested that wild-type (WT) Ras exerts tumor suppressor activity. Expressing oncogenic Nras[superscript G12D] in the hematopoietic compartment of mice induces an aggressive myeloproliferative neoplasm that is exacerbated in homozygous mutant animals. Here, we show that increased Nras[superscript G12D] gene dosage, but not inactivation of WT Nras, underlies the aggressive in vivo behavior of Nras[superscript G12D over G12D] hematopoietic cells. Modulating Nras[superscript G12D] dosage had discrete effects on myeloid progenitor growth, signal transduction, and sensitivity to MAP-ERK kinase (MEK) inhibition. Furthermore, enforced WT N-Ras expression neither suppressed the growth of Nras-mutant cells nor inhibited myeloid transformation by exogenous Nras[superscript G12D]. Importantly, NRAS expression increased in human cancer cell lines with NRAS mutations. These data have therapeutic implications and support reconsidering the proposed tumor suppressor activity of WT Ras in other cancers.Pfizer Inc. (PD0325901)National Institutes of Health (U.S.) (Grant R37CA72614)National Institutes of Health (U.S.) (Grant P01CA40046)National Institutes of Health (U.S.) (Grant K08CA134649)Leukemia & Lymphoma Society of America (Specialized Center of Research Award LLS 7019-04))American Lebanese Syrian Associated Charitie

Data from: Monoallelic loss of the imprinted gene Grb10 promotes tumor formation in irradiated Nf1+/- mice

Imprinted genes are expressed from only one parental allele and heterozygous loss involving the expressed allele is sufficient to produce complete loss of protein expression. Genetic alterations are common in tumorigenesis but the role of imprinted genes in this process is not well understood. In earlier work we mutagenized mice heterozygous for the Neurofibromatosis I tumor suppressor gene (NF1) to model radiotherapy-associated second malignant neoplasms that arise in irradiated NF1 patients. Expression analysis of tumor cell lines established from our mouse models identified Grb10 expression as widely absent. Grb10 is an imprinted gene and polymorphism analysis of cell lines and primary tumors demonstrates that the expressed allele is commonly lost in diverse Nf1 mutant tumors arising in our mouse models. We performed functional studies to test whether Grb10 restoration or loss alter fundamental features of the tumor growth. Restoring Grb10 in Nf1 mutant tumors decreases proliferation, decreases soft agar colony formation and downregulates Ras signaling. Conversely, Grb10 silencing in untransformed mouse embryo fibroblasts significantly increased cell proliferation and increased Ras-GTP levels. Expression of a constitutively activated MEK rescued tumor cells from Grb10-mediated reduction in colony formation. These studies reveal that Grb10 loss can occur during in vivo tumorigenesis, with a functional consequence in untransformed primary cells. In tumors, Grb10 loss independently promotes Ras pathway hyperactivation, which promotes hyperproliferation, an early feature of tumor development. In the context of a robust Nf1 mutant mouse model of cancer this work identifies a novel role for an imprinted gene in tumorigenesis

Grb10 sequence data

This file contains Grb10 exomic sequences from tumors described in the manuscript

<i>Grb10</i> expression reduces soft agar colony formation by <i>Nf1</i> mutant tumor cell line.

<p>A. Retro-virally expressed HA-Grb10 protein in 989 tumor cells was identified by immunoblotting for the HA tag. B. Soft agar colony formation assay quantified for each cell line. Data from 4 independent experiments, each with replicates of 4. (Student’s t-test, *** p<0.001, *p< 0.05) C. Photographs of representative plate stained for colonies from each group. D. 989 tumor cells expressing either wildtype or mutant Grb10 were serum starved for 18 hours, then stimulated with insulin (150 nM). Whole cell lysates of tumor cells were collected at 0, 5 and 15 minutes after insulin was added, then assessed by immunoblotting for phospho-specific antibodies against activated Ras effectors Akt Ser 473, ERK 42/44 Thr 202/Tyr204, and S6 Ser235/236. Corresponding Ras-GTP pull-down is shown on bottom line.</p

Expression of constitutively activated MEK rescues tumor cells from <i>Grb10</i> mediated suppression of colony formation.

<p>A. Flag-tagged Grb10, MEK DD, or both were expressed in 989 tumor cells. Expression was confirmed by immunoblotting for Flag and phosphorylated Akt and ERK were visualized. Actin is shown as loading control. B/C. 989 tumor cells expressing Flag-tagged Grb10, MEK DD, or both, as shown in panel (A), were grown in soft agar colony formation assay. Photographs of representative plates stained for colonies (B) and quantitation of colony formation (C) are shown.</p

<i>Grb10</i> chromosomal position, inheritance, and loss in <i>Nf1</i> mutant mouse tumors.

<p>A. Proportion of tumors showing LOH along chromosome 11 in <i>Nf1</i> mutant tumors. (Carcinomas n = 26, sarcomas n = 26, pheochromocytomas n = 52) B. Breeding schema describing inheritance of mutant and wildtype <i>Nf1</i> genes. C. Microsatellites assessed by PCR-based fragment analysis to verify LOH in <i>Grb10</i> gene and identify parental allele involved (BL6 or 129). (**) indicates loss. Reduced C57Bl/6-derived peak in <i>Nf1</i> mutant cell lines 930, 989, 9223, indicating LOH of maternal C57Bl/6 allele, while cell line 867 has loss of the paternal 129-derived allele. D. Schematic of predominant pattern of <i>Grb10</i> and <i>Nf1</i> loss.</p

<i>Grb10</i> expression in diverse tumors suppresses proliferation.

<p>A. Wildtype <i>Grb10</i> or GFP was expressed in sarcoma line 963, which is wildtype for <i>Nf1</i>. <i>Grb10</i> restoration was confirmed by immunoblotting for the FLAG tag, and pAKT and pERK levels were assessed. B. Cell proliferation was assessed 4 days after plating and was reduced in 963 tumor cells after <i>Grb10</i> restoration (t-test, **p<0.01). C. Wildtype <i>Grb10</i> or GFP was expressed in ^<i>V16</i>HRas-transformed human astrocytes and 881 cells for comparison. <i>Grb10</i> overexpression was confirmed by immunoblotting for Flag, and phosphorylated and total AKT and ERK were assessed. To visualize the markedly different levels of <i>Grb10</i> expression between the cell lines, two exposures (3 minutes and 1 minute) are shown. D. Cell proliferation was assessed 4 days after plating was significantly reduced in 881 (null for <i>Nf1</i>) and ^<i>V16</i>HRas-transformed human astrocytes expressing <i>Grb10</i> (t-test, **p<0.01). E. The <i>NF1</i> mutant human tumor cell lines 02.2, 94.3, 96.2 were assessed for total Grb10 protein, and phosphorylated AKT and ERK by immunoblotting. F. GFP or FLAG-tagged wildtype <i>Grb10</i> was expressed in the human <i>NF1</i> mutant tumor cell line 02.2. Cell lysates were immunoblotted for the FLAG tag, phosphorylated AKT and ERK, total AKT and ERK, and Actin. G. 02.2 cells expressing either GFP or Grb10 were compared for differences in cell proliferation rates after 4 days in culture (t-test, **<0.01). The histogram is representative of 1 of 3 experiments.</p