Lynn S. White

Absence of Apparent Phenotype in Mice Lacking Cdc25C Protein Phosphatase

ABSTRACT The Cdc25 family of protein phosphatases positively regulate the cell division cycle by activating cyclin-dependent protein kinases. In humans and rodents, three Cdc25 family members denoted Cdc25A, -B, and -C have been identified. The murine forms of Cdc25 exhibit distinct patterns of expression both during development and in adult mouse tissues. In order to determine unique contributions made by the Cdc25C protein phosphatase to embryonic and adult cell cycles, mice lacking Cdc25C were generated. We report thatCdc25C−/− mice are viable and do not display any obvious abnormalities. Among adult tissues in whichCdc25C is detected, its transcripts are most abundant in testis, followed by thymus, ovary, spleen, and intestine. Mice lackingCdc25C were fertile, indicating that Cdc25Cdoes not contribute an essential function during spermatogenesis or oogenesis in the mouse. T- and B-cell development was also found to be normal in Cdc25C −/− mice, andCdc25C −/− mouse splenic T and B cells exhibited normal proliferative responses in vitro. Finally, the phosphorylation status of Cdc2, the timing of entry into mitosis, and the cellular response to DNA damage were unperturbed in mouse embryo fibroblasts lacking Cdc25C. These findings indicate thatCdc25A and/or Cdc25B may compensate for loss ofCdc25C in the mouse.

CD45 regulates Src family member kinase activity associated with macrophage integrin-mediated adhesion

BACKGROUND Adhesion of leukocytes to the extracellular matrix and to other cells is mediated by members of the integrin family of adhesion molecules. Src family kinases are activated upon integrin-mediated adhesion. In lymphocytes, CD45 is a leukocyte-specific transmembrane protein tyrosine phosphatase that activates Src family kinases associated with B-cell and T-cell antigen receptor signaling by constitutive dephosphorylation of the inhibitory carboxy-terminal tyrosine phosphorylation site. Here, we show that CD45 is also important in downregulating the kinase activity of Src family members during integrin-mediated adhesion in macrophages. RESULTS We found that CD45 colocalized with beta2 integrin and the Src family kinase p53/56(lyn) to adhesion sites in bone marrow-derived macrophages. Macrophages from CD45(-/-) mice were unable to maintain integrin-mediated adhesion. In adherent macrophages, absence of CD45 led to the hyperphosphorylation and hyperactivation of p56/59(hck) and p53/56(lyn), but not of p58(c-fgr). CD45 directly inactivated p59(hck) but not p56(lck) in transient transfection assays. Furthermore, coexpression of CD45 with p59(hck) or p56(lyn) containing a tyrosine to phenylalanine mutation at the carboxy-terminal negative regulatory site resulted in decreased tyrosine phosphorylation of the Src family member kinases due to dephosphorylation of the potentiating tyrosine phosphorylation site within the kinase domain. CONCLUSIONS Using primary bone marrow macrophages, these studies demonstrate that CD45 regulates Src family kinases and is required to maintain macrophage adhesion. CD45 decreases Src family kinase activity by dephosphorylating the tyrosine residue located within the kinase domain.

Normal Cell Cycle and Checkpoint Responses in Mice and Cells Lacking Cdc25B and Cdc25C Protein Phosphatases

ABSTRACT The Cdc25 family of protein phosphatases positively regulates cell division by activating cyclin-dependent protein kinases (CDKs). In humans and rodents, there are three Cdc25 family members—denoted Cdc25A, Cdc25B, and Cdc25C—that can be distinguished based on their subcellular compartmentalizations, their abundances and/or activities throughout the cell cycle, the CDKs that they target for activation, and whether they are overexpressed in human cancers. In addition, murine forms of Cdc25 exhibit distinct patterns of expression throughout development and in adult tissues. These properties suggest that individual Cdc25 family members contribute distinct biological functions in embryonic and adult cell cycles of mammals. Interestingly, mice with Cdc25C disrupted are healthy, and cells derived from these mice exhibit normal cell cycles and checkpoint responses. Cdc25B− / − mice are also generally normal (although females are sterile), and cells derived from Cdc25B − / − mice have normal cell cycles. Here we report that mice lacking both Cdc25B and Cdc25C are obtained at the expected Mendelian ratios, indicating that Cdc25B and Cdc25C are not required for mouse development or mitotic entry. Furthermore, cell cycles, DNA damage responses, and Cdc25A regulation are normal in cells lacking Cdc25B and Cdc25C. These findings indicate that Cdc25A, or possibly other phosphatases, is able to functionally compensate for the loss of Cdc25B and Cdc25C in mice.

Immune System Dysfunction and Autoimmune Disease in Mice Lacking Emk (Par-1) Protein Kinase

ABSTRACT Emk is a serine/threonine protein kinase implicated in regulating polarity, cell cycle progression, and microtubule dynamics. To delineate the role of Emk in development and adult tissues, mice lacking Emk were generated by targeted gene disruption.Emk−/− mice displayed growth retardation and immune cell dysfunction. Although B- and T-cell development were normal, CD4+T cells lacking Emk exhibited a marked upregulation of the memory marker CD44/pgp-1 and produced more gamma interferon and interleukin-4 on stimulation through the T-cell receptor in vitro. In addition, B-cell responses to T-cell-dependent and -independent antigen challenge were altered in vivo. AsEmk −/− animals aged, they developed splenomegaly, lymphadenopathy, membranoproliferative glomerulonephritis, and lymphocytic infiltrates in the lungs, parotid glands and kidneys. Taken together, these results demonstrate that the Emk protein kinase is essential for maintaining immune system homeostasis and that loss of Emk may contribute to autoimmune disease in mammals.

The protein tyrosine phosphatase SHP-1 regulates integrin-mediated adhesion of macrophages

The Src homology 2 domain phosphatase-1 (SHP-1) is a tyrosine phosphatase containing two amino-terminal SH2 domains and is expressed primarily by hematopoietic-derived cells [1]. The viable motheaten (Hcphme-v) mutant mice (mev) suffer from progressive inflammation due to a deficiency of SHP-1 enzyme activity [2,3] and die at 3-4 months of age from macrophage and neutrophil accumulation in the lung [4]. The mechanism by which SHP-1 deficiency leads to inflammation is unknown. We found that macrophages from mev mice adhered and spread to a greater extent than normal macrophages through alpha m beta 2 integrin-mediated contacts. Whereas macrophages deficient in the transmembrane tyrosine phosphatase CD45 (CD45-/-) spontaneously detached from alpha m beta 2 integrin contacts [5], cells deficient in both CD45 and SHP-1 did not. In SHP-1 deficient macrophages there was a 10-15-fold increase in D-3 phospholipid products of phosphatidylinositol (PI) 3-kinase. Concomitantly, there was a 2-5-fold increase in membrane-associated PI 3-kinase activity in mev macrophages relative to normal macrophages. Treatment of macrophages with the PI 3-kinase inhibitors wortmannin or LY294002 resulted in a dramatic detachment of cells, indicating that PI 3-kinase activity is required for adhesion. These data demonstrate that SHP-1 is necessary for detachment from alpha m beta 2 integrin-mediated contacts in primary macrophages and suggest that a defect in this pathway may contribute to inflammatory disease.

Response of small intestinal epithelial cells to acute disruption of cell division through CDC25 deletion

The CDC25 protein phosphatases (CDC25A, B, and C) drive cell cycle transitions by activating key components of the cell cycle engine. CDC25A and CDC25B are frequently overproduced in human cancers. Disruption of Cdc25B or Cdc25C individually or in combination has no effect on mouse viability. Here we report that CDC25A is the only family member to provide an essential function during early embryonic development, and that other family members compensate for its loss in adult mice. In contrast, conditional disruption of the entire family is lethal in adults due to a loss of small intestinal epithelial cell proliferation in crypts of Lieberkühn. Cdc25 loss induced Wnt signaling, and overall crypt structures were preserved. In the face of continuous Wnt signaling, nearly all crypt epithelial progenitors differentiated into multiple cell lineages, including crypt base columnar cells, a proposed stem cell. A small population of Musashi/Dcamkl-1/nuclear β-catenin–positive epithelial cells was retained in these crypts. These findings have implications for the development of novel, less cytotoxic cancer chemotherapeutic drugs that specifically target the cell cycle.

Loss of the Par-1b/MARK2 polarity kinase leads to increased metabolic rate, decreased adiposity, and insulin hypersensitivity in vivo

Obesity is a major factor central to the development of insulin resistance and type 2 diabetes. The identification and characterization of genes involved in regulation of adiposity, insulin sensitivity, and glucose uptake are key to the design and development of new drug therapies for this disease. In this study, we show that the polarity kinase Par-1b/MARK2 is required for regulating glucose metabolism in vivo. Mice null for Par-1b were lean, insulin hypersensitive, resistant to high-fat diet-induced weight gain, and hypermetabolic. 18F-FDG microPET and hyperinsulinemic–euglycemic clamp analyses demonstrated increased glucose uptake into white and brown adipose tissue, but not into skeletal muscle of Par-1b null mice relative to wild-type controls. Taken together, these data indicate that Par-1b is a regulator of glucose metabolism and adiposity in the whole animal and may be a valuable drug target for the treatment of both type 2 diabetes and obesity.

Restoration of Thymocyte Development and Function in zap-70−/− Mice by the Syk Protein Tyrosine Kinase

The Syk family of protein tyrosine kinases, consisting of ZAP-70 and Syk, associate with the pre- and alphabeta T cell antigen receptors (TCRs) and undergo tyrosine phosphorylation and activation following receptor engagement. Thymocyte development in zap-70-/- mice is blocked at the CD4+CD8+ TCR(lo) stage. The presence of Syk in the thymus has raised the possibility that Syk may be able to mediate TCR function. To determine if Syk can play a role in thymocyte development, we generated zap-70-/- mice expressing a human syk cDNA. Syk expression restored both thymocyte development and function. In addition, Syk function required the CD45 transmembrane protein tyrosine phosphatase. Hence, ZAP-70 and Syk can play overlapping functions and exhibit similar regulatory mechanisms in mediating alphabeta T cell development.

Synthetic lethality of Chk1 inhibition combined with p53 and/or p21 loss during a DNA damage response in normal and tumor cells

Cell cycle checkpoints ensure genome integrity and are frequently compromised in human cancers. A therapeutic strategy being explored takes advantage of checkpoint defects in p53-deficient tumors in order to sensitize them to DNA-damaging agents by eliminating Chk1-mediated checkpoint responses. Using mouse models, we demonstrated that p21 is a key determinant of how cells respond to the combination of DNA damage and Chk1 inhibition (combination therapy) in normal cells as well as in tumors. Loss of p21 sensitized normal cells to the combination therapy much more than did p53 loss and the enhanced lethality was partially blocked by CDK inhibition. In addition, basal pools of p21 (p53 independent) provided p53 null cells with protection from the combination therapy. Our results uncover a novel p53-independent function for p21 in protecting cells from the lethal effects of DNA damage followed by Chk1 inhibition. As p21 levels are low in a significant fraction of colorectal tumors, they are predicted to be particularly sensitive to the combination therapy. Results reported in this study support this prediction.

Loss of Par-1a/MARK3/C-TAK1 kinase leads to reduced adiposity, resistance to hepatic steatosis, and defective gluconeogenesis

ABSTRACT Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a−/− but not in control or Par-1b−/− mice. The intercrossing of Par-1a−/− with Par-1b−/− mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a−/− mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b−/− mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice.