Yulia Artemenko

Moving towards a paradigm: common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes

Chemotaxis, or directed migration of cells along a chemical gradient, is a highly coordinated process that involves gradient sensing, motility, and polarity. Most of our understanding of chemotaxis comes from studies of cells undergoing amoeboid-type migration, in particular the social amoeba Dictyostelium discoideum and leukocytes. In these amoeboid cells the molecular events leading to directed migration can be conceptually divided into four interacting networks: receptor/G protein, signal transduction, cytoskeleton, and polarity. The signal transduction network occupies a central position in this scheme as it receives direct input from the receptor/G protein network, as well as feedback from the cytoskeletal and polarity networks. Multiple overlapping modules within the signal transduction network transmit the signals to the actin cytoskeleton network leading to biased pseudopod protrusion in the direction of the gradient. The overall architecture of the networks, as well as the individual signaling modules, is remarkably conserved between Dictyostelium and mammalian leukocytes, and the similarities and differences between the two systems are the subject of this review.

Anti-adipogenic effect of PDGF is reversed by PKC inhibition.

Healthy adipose tissue function depends on adipogenesis. The capacity to form new adipocytes prevents the emergence of insulin‐resistant hypertrophied adipocytes, as well as the deleterious lipid deposition in muscle, liver, and pancreas. It is therefore important to understand how adipogenesis is modulated. Platelet‐derived growth factor (PDGF) is anti‐adipogenic, but the stage of differentiation that it targets, and the signaling pathways that it triggers, are not defined. We have studied the inhibitory effect of PDGF on murine 3T3‐L1 preadipocyte and human preadipocyte differentiation. There was a significant attenuation in the protein expression of the adipogenic transcription factors, PPARγ and C/EBPα, as well as in the levels of later differentiation markers, including adiponectin, aP2, and fatty acid synthase. PDGF treatment resulted in the persistence of PDGF receptor and PKCα expression, in contrast to the expected downregulation of both proteins that occurs during differentiation. Inactivation of conventional PKC isoforms, by bisindolylmaleimide I or PKC pseudosubstrate M20–28, partially reversed the inhibition of 3T3‐L1 and human preadipocyte differentiation by PDGF, as assessed by fatty acid synthase expression and morphological appearance.

Tumor suppressor Hippo/MST1 kinase mediates chemotaxis by regulating spreading and adhesion

Chemotaxis depends on a network of parallel pathways that coordinate cytoskeletal events to bias cell movement along a chemoattractant gradient. Using a forward genetic screen in Dictyostelium discoideum, we identified the Ste20 kinase KrsB, a homolog of tumor suppressors Hippo and MST1/2, as a negative regulator of cell spreading and substrate attachment. The excessive adhesion of krsB− cells reduced directional movement and prolonged the streaming phase of multicellular aggregation. These phenotypes depended on an intact kinase domain and phosphorylation of a conserved threonine (T176) within the activation loop. Chemoattractants triggered a rapid, transient autophosphorylation of T176 in a heterotrimeric G protein-dependent and PI3K- and TorC2-independent manner. The active phosphorylated form of KrsB acts to decrease adhesion to the substrate. Taken together these studies suggest that cycling between active and inactive forms of KrsB may provide the dynamic regulation of cell adhesion needed for proper cell migration and chemotaxis. KrsB interacts genetically with another D. discoideum Hippo/MST homolog, KrsA, but the two genes are not functionally redundant. These studies show that Hippo/MST proteins, like the tumor suppressor PTEN and oncogenes Ras and PI3K, play a key role in cell morphological events in addition to their role in regulating cell growth.

Regulation of endogenous SH2 domain-containing inositol 5-phosphatase (SHIP2) in 3T3-L1 and human preadipocytes

The role of the inositol lipid 5‐phosphatase (SHIP2) in preadipocyte signaling is not known. Although overexpression of SHIP2 inhibited proliferation and 3H‐thymidine incorporation in 3T3‐L1 preadipocytes, there was no effect on insulin‐induced adipogenesis. Insulin promoted SHIP2 tyrosine phosphorylation in differentiated 3T3‐L1 adipocytes, but did not do so in preadipocytes. The absence of SHIP2 tyrosine phosphorylation suggests a potential explanation for the isolated rise in PI(3,4,5)P3, without any changes in PI(3,4)P2, previously observed following insulin treatment of these cells. Lack of SHIP2 tyrosine phosphorylation by insulin was also observed in primary cultures of human abdominal subcutaneous preadipocytes. These cells also produced PI(3,4,5)P3, but not PI(3,4)P2, in response to insulin. Comparison of insulin vs. PDGF treatment on SHIP2 tyrosine phosphorylation in 3T3‐L1 and human preadipocytes revealed that only PDGF, which stimulates the accumulation of PI(3,4,5)P3 as well as PI(3,4)P2, was active in this regard, and only PDGF promoted the association of 52 kDa form of Shc with SHIP2. Nevertheless, both insulin and PDGF were equally effective in translocating SHIP2 to the plasma membrane in 3T3‐L1 preadipocytes. Lack of SHIP2 tyrosine phosphorylation may account for the insulin‐specific inositol phospholipid pattern of accumulation in preadipocytes. J. Cell. Physiol. 197: 243–250, 2003© 2003 Wiley‐Liss, Inc.

Regulation of PDGF-stimulated SHIP2 tyrosine phosphorylation and association with Shc in 3T3-L1 preadipocytes.

In 3T3‐L1 and human preadipocytes, insulin results in the isolated rise in phosphatidylinositol (PI)‐3,4,5‐P3, whereas PDGF produces PI(3,4)P2 in addition to PI(3,4,5)P3. SH2 domain‐containing inositol 5‐phosphatase 2 (SHIP2) converts PI(3,4,5)P3 into PI(3,4)P2. PDGF, but not insulin, stimulates SHIP2 tyrosine phosphorylation and its association with Shc in human and 3T3‐L1 preadipocytes. We now demonstrate that SHIP2 tyrosine phosphorylation and association with Shc in PDGF‐treated 3T3‐L1 preadipocytes was reduced by bisindolylmaleimide I (BisI), an inhibitor of conventional/novel protein kinase C (PKC). However, the production of PI(3,4)P2 and PI(3,4,5)P3 by PDGF was unaffected by BisI. Activation of PKC by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was not sufficient to induce SHIP2 tyrosine phosphorylation. Furthermore, we identified threonine 958 (T958) as a novel PDGF‐responsive SHIP2 phosphorylation site. Mutation of T958 to alanine reduced PDGF‐stimulated SHIP2 tyrosine phosphorylation and association with Shc, but did not alter its anti‐proliferative effect on preadipocytes. This study demonstrates that SHIP2 tyrosine phosphorylation and Shc association can be regulated by serine/threonine signaling pathways, either indirectly (via PKC), or directly (via T958). Interestingly, the anti‐proliferative effect of SHIP2 T958A, as well as another SHIP2 mutant (Y986F, Y987F) that also displays defective tyrosine phosphorylation and Shc association, does not depend on these molecular events. J. Cell. Physiol. 211: 598–607, 2007.

Catalytically inactive SHIP2 inhibits proliferation by attenuating pdgf signaling in 3T3‐L1 preadipocytes

Inadequate proliferation and/or differentiation of preadipocytes may lead to adipose tissue dysfunction characterized by hypertrophied, insulin‐resistant adipocytes. Platelet‐derived growth factor (PDGF) may alter adipose tissue function by promoting proliferation of preadipocytes. Two principal signaling pathways that regulate proliferation are PI3K/PI(3,4,5)P3/Akt and Shc/Ras/ERK1/2. SH2 domain‐containing inositol 5‐phosphatase 2 (SHIP2) dephosphorylates PI(3,4,5)P3, and also binds to Shc. Our goal was to determine how SHIP2 affects these PDGF signaling routes. To assess the role of the 5‐phosphatase domain, we expressed wild‐type or catalytically inactive dominant‐negative SHIP2 (P686A‐D690A‐R691A; PDR/AAA) in 3T3‐L1 preadipocytes. Surprisingly, SHIP2 PDR/AAA inhibited proliferation more potently than wild‐type SHIP2. After three days of proliferation, phospho‐Akt, phospho‐ERK1/2, and PDGF receptor (PDGFR) levels were reduced in PDR/AAA‐expressing preadipocytes. SHIP2 PDR/AAA interference with PDGFR signaling was demonstrated using imatinib, an inhibitor of PDGFR tyrosine kinase. The anti‐proliferative effect of imatinib observed in control preadipocytes was not significant in SHIP2 PDR/AAA‐expressing preadipocytes, indicating a pre‐existing impairment of PDGFR‐dependent mitogenesis in these cells. The inhibition of PDGF‐activated mitogenic pathways by SHIP2 PDR/AAA was consistent with a decrease in PDGFR phosphorylation caused by a drop in receptor levels in SHIP2 PDR/AAA‐expressing cells. SHIP2 PDR/AAA promoted ubiquitination of the PDGFR and its degradation via the lysosomal pathway independently of the association between the E3 ubiquitin ligase c‐Cbl and PDGFR. Overall, our findings indicate that SHIP2 PDR/AAA reduces preadipocyte proliferation by attenuating PDGFR signaling. J. Cell. Physiol. 218: 228–236, 2009.

Chemical and mechanical stimuli act on common signal transduction and cytoskeletal networks

Significance Cells directionally migrate in response to a variety of external cues, including chemical, electrical, and mechanical stimuli; however, only response to chemoattractants has been characterized at the molecular level. Binding of chemoattractants to specific surface receptors triggers rapid, transient activation of many signal transduction and cytoskeletal events. We discovered that brief application of shear stress to cells likely elicits activation of all of the same events. Responses to chemoattractants and shear stress are susceptible to many of the same perturbations, although that to mechanical stimulation uniquely is blocked by disruption of the actin cytoskeleton. Our finding provides insight into the molecular mechanism of cellular response to mechanical stimuli and has important implications for integration of chemical and mechanical inputs. Signal transduction pathways activated by chemoattractants have been extensively studied, but little is known about the events mediating responses to mechanical stimuli. We discovered that acute mechanical perturbation of cells triggered transient activation of all tested components of the chemotactic signal transduction network, as well as actin polymerization. Similarly to chemoattractants, the shear flow-induced signal transduction events displayed features of excitability, including the ability to mount a full response irrespective of the length of the stimulation and a refractory period that is shared with that generated by chemoattractants. Loss of G protein subunits, inhibition of multiple signal transduction events, or disruption of calcium signaling attenuated the response to acute mechanical stimulation. Unlike the response to chemoattractants, an intact actin cytoskeleton was essential for reacting to mechanical perturbation. These results taken together suggest that chemotactic and mechanical stimuli trigger activation of a common signal transduction network that integrates external cues to regulate cytoskeletal activity and drive cell migration.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.