Phosphorylation of the Unique C-Terminal Tail of the Alpha Isoform of the Scaffold Protein SH2B1 Controls the Ability of SH2B1α To Enhance Nerve Growth Factor Function.

The scaffold protein SH2B1, a major regulator of body weight, is recruited to the receptors of multiple cytokines and growth factors, including nerve growth factor (NGF). The ß isoform but not the α isoform of SH2B1 greatly enhances NGF-dependent neurite outgrowth of PC12 cells. Here, we asked how the unique C-terminal tails of the α and ß isoforms modulate SH2B1 function. We compared the actions of SH2B1α and SH2B1ß to those of the N-terminal 631 amino acids shared by both isoforms. In contrast to the ß tail, the α tail inhibited the ability of SH2B1 to both cycle through the nucleus and enhance NGF-mediated neurite outgrowth, gene expression, phosphorylation of Akt and phospholipase C-gamma (PLC-γ), and autophosphorylation of the NGF receptor TrkA. These functions were restored when Tyr753 in the α tail was mutated to phenylalanine. We provide evidence that TrkA phosphorylates Tyr753 in SH2B1α, as well as tyrosines 439 and 55 in both SH2B1α and SH2B1ß. Finally, coexpression of SH2B1α but not SH2B1α with a mutation of Y to F at position 753 (Y753F) inhibited the ability of SH2B1ß to enhance neurite outgrowth. These results suggest that the C-terminal tails of SH2B1 isoforms are key determinants of the cellular role of SH2B1. Furthermore, the function of SH2B1α is regulated by phosphorylation of the α tail.

SOX9 Regulates Cancer Stem-Like Properties and Metastatic Potential of Single-Walled Carbon Nanotube-Exposed Cells.

Engineered nanomaterials hold great promise for the future development of innovative products but their adverse health effects are a major concern. Recent studies have indicated that certain nanomaterials, including carbon nanotubes (CNTs), may be carcinogenic. However, the underlying mechanisms behind their potential malignant properties remain unclear. In this study, we linked SOX9, a stem cell associated transcription factor, to the neoplastic-like properties of human lung epithelial cells chronically exposed to a low-dose of single-walled carbon nanotubes (SWCNTs). We found that SOX9 is upregulated in SWCNT-exposed cells, which is consistent with their abilities to induce tumor formation and metastasis in vivo. We therefore hypothesized that SOX9 overexpression may be responsible for the neoplastic-like phenotype observed in our model. Indeed, SOX9 knockdown inhibited anchorage-independent cell growth in vitro and lung colonization in vivo in a mouse xenograft model. SOX9 depletion also suppressed the formation of cancer stem-like cells (CSCs), as determined by tumor sphere formation and aldehyde dehydrogenase (ALDH) activity (Aldefluor) assays. Furthermore, SOX9 knockdown suppressed tumor metastasis and the expression of the stem cell marker ALDH1A1. Taken together, our findings provide a mechanistic insight into SWCNT-induced carcinogenesis and the role of SOX9 in CSC regulation and metastasis.

Induction of Slug by Chronic Exposure to Single-Walled Carbon Nanotubes Promotes Tumor Formation and Metastasis.

Carbon nanotubes (CNTs) represent a major class of engineered nanomaterials that are being used in diverse fields. However, their use has increasingly become a concern because of their carcinogenic potential. Accumulating evidence has demonstrated that certain types of CNTs are carcinogenic or tumor-promoting in animal models. However, the underlying molecular and cellular mechanisms are unclear. Here, we report that chronic exposure to single-walled (SW) CNTs results in the induction of Slug, a key transcription factor that induces an epithelial-mesenchymal transition (EMT), in human lung epithelial cells. We show that SWCNT-induced Slug upregulation plays a critical role in the aggressive phenotype of SWCNT-exposed cells, which includes increased cell migration, invasion, and anchorage-independent cell growth. Our in vivo studies also show that SWCNT-induced Slug upregulation and EMT activation play a pivotal role in tumor formation and metastasis. Our findings illustrate a direct link between CNT-induced Slug upregulation, EMT activation, and tumor formation and metastasis, and they highlight the potential of CNT-induced Slug upregulation as a target for future risk assessment and prevention of CNT-associated diseases.

Mesothelin promotes epithelial-to-mesenchymal transition and tumorigenicity of human lung cancer and mesothelioma cells.

BACKGROUND: Lung cancer and pleural mesothelioma are two of the most deadly forms of cancer. The prognosis of lung cancer and mesothelioma is extremely poor due to limited treatment modalities and lack of understanding of the disease mechanisms. We have identified mesothelin as a potentially unique therapeutic target that as a specific advantage appears nonessential in most cell types. Mesothelin (MSLN), a plasma membrane differentiation antigen, is expressed at a high level in many human solid tumors, including 70% of lung cancer and nearly all mesotheliomas. However, the role of MSLN in the disease process and underlying mechanisms is largely unknown. METHODS: ShRNA knockdown and overexpression of MSLN were performed in human cancer cell lines and corresponding normal cells, respectively. Tumorigenic and metastatic effects of MSLN were examined by tumor sphere formation, migration, and invasion assays in vitro, as well as xenograft tumor assay in vivo. EMT and CSCs were detected by qPCR array, immunoblotting and flow cytometry. RESULTS: MSLN plays a key role in controlling epithelial-to-mesenchymal transition (EMT) and stem properties of human lung cancer and mesothelioma cells that control their tumorigenicity and metastatic potential. Firstly, MSLN was found to be highly upregulated in non-small cell lung cancer (NSCLC) patient tissues and in lung carcinoma and mesothelioma cell lines. Secondly, genetic knockdown of MSLN significantly reduced anchorage-independent cell growth, tumor sphere formation, cell adhesion, migration and invasion in vitro, as well as tumor formation and metastasis in vivo. Thirdly, ectopic overexpression of MSLN induced the malignant phenotype of non-cancerous cells, supporting its role as an oncogene. Finally, mechanistic studies revealed that knockdown of MSLN reversed EMT and attenuated stem cell properties, in addition to inhibiting tumor growth and metastasis. CONCLUSIONS: These results indicate an essential role of MSLN in controlling EMT and stem cell properties of human lung cancer and mesothelioma cells. Since EMT is an important process in tumor progression and metastasis, and MSLN is nonessential in most normal tissue, our findings on MSLN may provide new insights into the disease mechanisms and may aid in the development of novel targeted therapy for lung cancer and mesothelioma.

Problem-Based Learning: Cervical Adenocarcinoma Three-Session Case for First-Year Medical Students-Patient Minnie Pauls.

INTRODUCTION: Cervical adenocarcinoma represents a critical health problem in many underserved regions of the world and parts of the U.S. This module provides learning opportunities in the areas of female anatomy, physiology, histology, and pathology. This includes diagnosis by ultrasound and CT/PET scan, detailed staging and treatment of the cancer by various criteria, and future prevention by vaccination and screening. METHODS: Authors include a fourth-year medical student and a seasoned PBL facilitator with a basic science interest in cancer. In this problem-based learning module (PBL), a group of first-year medical students review the material that is released online for each of three weekly 90-minute sessions. Key learning issues are identified, researched out-of-class, and discussed at the beginning of the subsequent session. A differential diagnosis is weighed and the module culminates with a concept map drawn by students to integrate all relevant aspects and mechanisms of the case. RESULTS: The module was implemented twice with a small group of seven students. Students learned to correlate relevant biochemical mechanisms, histological, and anatomical features with the clinical signs and symptoms, to diagnose and suggest treatment options. The module was well-liked, and revised for publication by rebalancing the material based on specific student feedback. DISCUSSION: The PBL small-group format provides a unique opportunity over both semesters for first-year medical students to study clinical cases in a student-directed fashion and develop professional skills at various levels. Potential pitfalls lie in the online format, as this requires clear rules on computer usage and data sharing.

Carcinogenic Potential of High Aspect Ratio Carbon Nanomaterials.

Engineered nanomaterials, including high aspect ratio carbon nanomaterials, are often commercialized without a complete human risk assessment and safety evaluation. A health concern has been raised that high aspect ratio nanomaterials such as carbon nanotubes may cause unintended health consequences, such as asbestos-like lung cancer and mesothelioma, when chronically inhaled. Considering the widespread industrial and clinical applications and the increasing incidence of human exposure to nanomaterials, it is important to address the issue of nanomaterial carcinogenicity in a timely manner. This review summarizes recent advances in nanomaterial genotoxicity and carcinogenicity with a focus on high aspect ratio carbon nanotubes, and discusses current knowledge gaps and future research directions.

Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model.

In vitro disease modeling based on induced pluripotent stem cells (iPSCs) provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID), a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK) activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(D)J recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications.

Insulin receptor kinase-independent signaling via tyrosine phosphorylation of phosphatase PHLPP1.

Most insulin responses correlate well with insulin receptor (IR) Tyr kinase activation; however, critical exceptions to this concept have been presented. Specific IR mutants and stimulatory IR antibodies demonstrate a lack of correlation between IR kinase activity and specific insulin responses in numerous independent studies. IR conformation changes in response to insulin observed with various IR antibodies define an IR kinase-independent signal that alters the C-terminus. IR-related receptors in lower eukaryotes that lack a Tyr kinase point to an alternative mechanism of IR signaling earlier in evolution. However, the implied IR kinase-independent signaling mechanism remained obscure at the molecular level. Here we begin to define the molecular basis of an IR-dependent but IR kinase-independent insulin signal that is equally transmitted by a kinase-inactive mutant IR. This insulin signal results in Tyr phosphorylation and catalytic activation of phosphatase PHLPP1 via a PI 3-kinase-independent, wortmannin-insensitive signaling pathway. Dimerized SH2B1/PSM is a critical activator of the IR kinase and the resulting established insulin signal. In contrast it is an inhibitor of the IR kinase-independent insulin signal and disruption of SH2B1/PSM dimer binding to IR potentiates this signal. Dephosphorylation of Akt2 by PHLPP1 provides an alternative, SH2B1/PSM-regulated insulin-signaling pathway from IR to Akt2 of opposite polarity and distinct from the established PI 3-kinase-dependent signaling pathway via IRS proteins. In combination, both pathways should allow the opposing regulation of Akt2 activity at two phosphorylation sites to specifically define the insulin signal in the background of interfering Akt-regulating signals, such as those controlling cell proliferation and survival.

Mitogenic roles of Gab1 and Grb10 as direct cellular partners in the regulation of MAP kinase signaling.

Functions of signaling mediators Grb10 or Gab1 have been described in mitogenesis but remained disconnected. Here, we report the peptide hormone-dependent direct association between Grb10 and Gab1 and their functional connection in mitogenic signaling via MAP kinase using cultured fibroblasts as a model. In response to PDGF-, IGF-I, or insulin increased levels of Grb10 potentiated cell proliferation or survival whereas dominant-negative, domain-specific Grb10 peptide mimetics attenuated cell proliferation. This response was sensitive to p44/42 MAPK inhibitor but not to p38 MAPK inhibitor. In response to IGF-I or insulin Raf-1, MEK 1/2, and p44/42 MAPK were regulated by Grb10 but not Ras or p38 MAPK. In response to PDGF MEK 1/2, p44/42 MAPK and p38 MAPK were regulated by Grb10 but not Ras or Raf-1. Peptide hormone-dependent co-immunoprecipitation of Grb10 and Gab1 was demonstrated and specifically blocked by a Grb10 SH2 domain peptide mimetic. This domain was sufficient for direct, peptide hormone-dependent association with Gab1 via the Crk binding region. In response to PDGF, IGF-I, or insulin, in a direct comparison, elevated levels of mouse Grb10 delta, or human Grb10 beta or zeta equally potentiated fibroblast proliferation. Proliferation was severely reduced by Gab1 gene disruption whereas an elevated Gab1 gene dose proportionally stimulated Grb10-potentiated cell proliferation. In conclusion, Gab1 and Grb10 function as direct binding partners in the regulation of the mitogenic MAP kinase signal. In cultured fibroblasts, elevated levels of human Grb10 beta, zeta or mouse Grb10 delta comparably potentiate mitogenesis in response to PDGF, IGF-I, or insulin.

PSM/SH2B1 splice variants: critical role in src catalytic activation and the resulting STAT3s-mediated mitogenic response.

A role of PSM/SH2B1 had been shown in mitogenesis and extending to phenotypic cell transformation, however, the underlying molecular mechanism remained to be established. Here, four alternative PSM splice variants and individual functional protein domains were compared for their role in the regulation of Src activity. We found that elevated cellular levels of PSM variants resulted in phenotypic cell transformation and potentiated cell proliferation and survival in response to serum withdrawal. PSM variant activity presented a consistent signature pattern for any tested response of highest activity observed for gamma, followed by delta, alpha, and beta with decreasing activity. PSM-potentiated cell proliferation was sensitive to Src inhibitor herbimycin and PSM and Src were found in the same immune complex. PSM variants were substrates of the Src Tyr kinase and potentiated Src catalytic activity by increasing the V(max) and decreasing the K(m) for ATP with the signature pattern of variant activity. Dominant-negative PSM peptide mimetics including the SH2 or PH domains inhibited Src catalytic activity as well as Src-mediated phenotypic cell transformation. Activation of major Src substrate STAT3 was similarly potentiated by the PSM variants in a Src-dependent fashion or inhibited by PSM domain-specific peptide mimetics. Expression of a dominant-negative STAT3 mutant blocked PSM variant-mediated phenotypic cell transformation. Our results implicate an essential role of the PSM variants in the activation of the Src kinase and the resulting mitogenic response--extending to phenotypic cell transformation and involving the established Src substrate STAT3.

Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses.

The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin.

PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways.

The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.

Grb10: more than a simple adaptor protein.

Grb10 is a member of a superfamily of adaptor proteins that includes Grb7 and Grb14. This family of proteins shares a common overall structure, including an N-terminal region harboring a conserved proline-rich motif, a central Pleckstrin homology (PH) domain, a C-terminal Src homology 2 (SH2) domain, and a conserved region located between the PH and the SH2 domains (BPS). Grb10 directly interacts with a number of mitogenic receptor tyrosine kinases including the insulin (IR) and insulin-like growth factor-I (IGF-IR) receptor. Grb10 binds to the regulatory kinase loop of the insulin receptor (IR) via its SH2 and BPS domains. In addition to receptor tyrosine kinases, Grb10 has also been found to interact with non-receptor tyrosine kinases such as Tec and Bcr-Abl, and other cellular signaling molecules such as Raf-1 and the mitogen activated protein (MAP) kinase kinase, MEK. Overexpression of Grb10 has been shown to inhibit or stimulate insulin/IGF-I signaling depending on the expression levels of the specific isoforms, specific cell context, and/or physiologic endpoint. Genetic imprinting of Grb10 has been linked to the congenital disease, Silver-Russell syndrome, which is characterized by pre- and post-natal growth deficiency. This data suggests that Grb10 may function during embryogenesis in regulating insulin/IGF-I signaling as these growth factors play important roles during development. A role of Grb10 as a potent growth inhibitor during was implicated when disruption of the mGrb10 gene in mice resulted in overgrowth of mutant embryos and neonates. Grb10 is expressed in the central nervous system of mice and rats, which suggests that this protein may regulate neuronal insulin signaling and energy metabolism, consistent with its reported role in metabolic insulin action in fat and muscle cells. An important area of future investigation will be to elucidate the mechanism underlying Grb10's ability to regulate peptide hormone action including insulin/IGF-I signaling and to study the physiological role of this adaptor protein in cellular and animal models.

Grb10 exceeding the boundaries of a common signaling adapter.

Growth factor receptor binding protein 10 (Grb10) has been identified as a cellular partner of a number of receptor tyrosine kinases and other signaling mediators, compatible with multiple roles in mitogenic, metabolic, and embryogenic signaling that are also supported by the tissue distribution of Grb10. In particular, a role has been implicated in the regulation of PI 3-kinase signaling downstream of the insulin receptor. At least seven alternative splice variants have been identified within the Grb10 gene, a proposed candidate for some types of human Silver-Russell syndrome. Located on chromosome 7 (human) or 11 (mouse) the gene is oppositely imprinted in both species. Grb10 isoforms are members of a super family of signaling mediators that includes Grb7, Grb14, and Caenorhabditis elegans MIG-10. All mammalian members of this family share a domain structure which is represented by N-terminal (proline) Pro-rich sequences, a homology domain with MIG-10 (GM) which includes a Ras-associating (RA)-like domain, a pleckstrin homology region (PH), a C-terminal Src homology 2 (SH2) domain, and a receptor binding domain located between the PH and the SH2 domains termed BPS. Various Grb10 isoforms have been identified as cellular partners of the insulin receptor (IR) and insulin-like growth factor-I (IGF-I) receptor that provide the best-established regulators of Grb10 signaling. A regulatory role of Grb10 has been established in the respective metabolic and mitogenic responses by numerous lines of experimental evidence. However, the specific contribution of Grb10 was found to be highly dependent on the cellular context including the balance of other signaling mediators that define whether increased Grb10 levels will enhance or restrain a given response. This is supported by observations with super family members Grb7 and Grb14 that may engage in competitive and redundant mechanisms when compared to Grb10. Grb10 gene disruption in the mouse results in embryonal and placental overgrowth. The underlying molecular mechanisms and their interpretation remain open until a more comprehensive analysis will be available which includes the contribution of the Grb7 and Grb14 super family members. From a physiologic perspective at the cellular level increased levels of Grb10 have been shown to stimulate insulin metabolic action or mitogenic growth factor responses whereas peptide mimetics representing individual Grb10 domains were found to act oppositely by inhibiting the respective cellular response. In an alternative experimental context increased cellular levels of Grb10 have repeatedly been shown to inhibit cellular responses and signaling mechanisms. This has been most specifically observed at the level of molecular interactions in vitro. How the various observations relate to the physiologic role of cellular Grb10 remains to be established, also in the context of possible cross-talk to Grb14 and Grb7 signaling. Based on its interactions with a number of signaling mediators including protein kinases, adapters, and enzymes such as a ubiquitin ligase, Grb10 may act as a signaling hub to integrate multiple incoming signals and as a molecular scaffold to help assemble signaling complexes. The specific contribution of Grb10 in a signaling complex may depend on the local stochiometric balance of associating mediators, including the ratio of competing signaling proteins. In this context a constant cellular level of Grb10 may enhance or restrain a specific signaling mechanism depending on the local distribution and balance of specific Grb10 signaling partners. This concept is compatible with the diverse experimental observations on Grb10 function and emphasizes the importance of the specific cellular context to define the consequences of local changes in Grb10 distribution. Thus, to think of Grb10 as either a positive or negative signaling mediator will be inadequate in reflecting the complexity that underlies the final output of the Grb10 signal.

Growth factor receptor-binding protein 10 (Grb10) as a partner of phosphatidylinositol 3-kinase in metabolic insulin action.

The regulation of the metabolic insulin response by mouse growth factor receptor-binding protein 10 (Grb10) has been addressed in this report. We find mouse Grb10 to be a critical component of the insulin receptor (IR) signaling complex that provides a functional link between IR and p85 phosphatidylinositol (PI) 3-kinase and regulates PI 3-kinase activity. This regulatory mechanism parallels the established link between IR and p85 via insulin receptor substrate (IRS) proteins. A direct association was demonstrated between Grb10 and p85 but was not observed between Grb10 and IRS proteins. In addition, no effect of mouse Grb10 was observed on the association between IRS-1 and p85, on IRS-1-associated PI 3-kinase activity, or on insulin-mediated activation of IR or IRS proteins. A critical role of mouse Grb10 was observed in the regulation of PI 3-kinase activity and the resulting metabolic insulin response. Dominant-negative Grb10 domains, in particular the SH2 domain, eliminated the metabolic response to insulin in differentiated 3T3-L1 adipocytes. This was consistently observed for glycogen synthesis, glucose and amino acid transport, and lipogenesis. In parallel, the same metabolic responses were substantially elevated by increased levels of Grb10. A similar role of Grb10 was confirmed in mouse L6 cells. In addition to the SH2 domain, the Pro-rich amino-terminal region of Grb10 was implicated in the regulation of PI 3-kinase catalytic activity. These regulatory roles of Grb10 were extended to specific insulin mediators downstream of PI 3-kinase including PKB/Akt, glycogen synthase kinase, and glycogen synthase. In contrast, a regulatory role of Grb10 in parallel insulin response pathways including p70 S6 kinase, ubiquitin ligase Cbl, or mitogen-activated protein kinase p38 was not observed. The dissection of the interaction of mouse Grb10 with p85 and the resulting regulation of PI 3-kinase activity should help elucidate the complexity of the IR signaling mechanism.

Inhibition of protein kinase C catalytic activity by additional regions within the human protein kinase Calpha-regulatory domain lying outside of the pseudosubstrate sequence.

The N-terminal pseudosubstrate site within the protein kinase Calpha (PKCalpha)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCalpha-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940-8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCalpha-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Ralpha(39-177): this protein contained the full regulatory domain of human PKCalpha fused to glutathione S-transferase (GST), but lacked amino acids 1-38 (including the pseudosubstrate sequence) and amino acids 178-270 (including the C2 region)]. GST-Ralpha(39-177) significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCalpha holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33-86 within GST-Ralpha(39-177) dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159-242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of multiple regions within the PKCalpha-regulatory domain that play a direct role in the inhibition of catalytic domain activity.