Exploring the functional interaction between POSH and ALIX and the relevance to HIV-1 release.

BACKGROUND: The ALG2-interacting protein X (ALIX)/AIP1 is an adaptor protein with multiple functions in intracellular protein trafficking that plays a central role in the biogenesis of enveloped viruses. The ubiquitin E3-ligase POSH (plenty of SH3) augments HIV-1 egress by facilitating the transport of Gag to the cell membrane. Recently, it was reported, that POSH interacts with ALIX and thereby enhances ALIX mediated phenotypes in Drosophila. RESULTS: In this study we identified ALIX as a POSH ubiquitination substrate in human cells: POSH induces the ubiquitination of ALIX that is modified on several lysine residues in vivo and in vitro. This ubiquitination does not destabilize ALIX, suggesting a regulatory function. As it is well established that ALIX rescues virus release of L-domain mutant HIV-1, HIV-1DeltaPTAP, we demonstrated that wild type POSH, but not an ubiquitination inactive RING finger mutant (POSHV14A), substantially enhances ALIX-mediated release of infectious virions derived from HIV-1DeltaPTAP L-domain mutant (YPXnL-dependent HIV-1). In further agreement with the idea of a cooperative function of POSH and ALIX, mutating the YPXnL-ALIX binding site in Gag completely abrogated augmentation of virus release by overexpression of POSH. However, the effect of the POSH-mediated ubiquitination appears to be auxiliary, but not necessary, as silencing of POSH by RNAi does not disturb ALIX-augmentation of virus release. CONCLUSION: Thus, the cumulative results identified ALIX as an ubiquitination substrate of POSH and indicate that POSH and ALIX cooperate to facilitate efficient virus release. However, while ALIX is obligatory for the release of YPXnL-dependent HIV-1, POSH, albeit rate-limiting, may be functionally interchangeable.

Overexpression and forced activation of stat5 in mammary gland of transgenic mice promotes cellular proliferation, enhances differentiation, and delays postlactational apoptosis.

Signal transducer and activator of transcription 5 (Stat5) transduces extracellular cytokine and growth factor signals to the nucleus of mammary epithelial cells and thereby regulates gene transcription during pregnancy, lactation, and weaning. Gene constructs were prepared which subject the wild-type Stat5 or a constitutively active variant of Stat5 to the control of the beta-lactoglobulin (BLG) regulatory sequences and direct it to the mammary epithelium. The integrity and functionality of these constructs were confirmed through introduction into cultured mammary epithelial cells and hormone induction experiments. Expression levels and states of activity of Stat5 in mammary gland tissue were manipulated by introducing Stat5 variants as transgenes into the pronuclei of transgenic mice. The consequences of enhanced Stat5 expression and activation on the development of alveoli, their differentiated functions, and on postlactational involution were investigated. As expected, the transgenic mouse lines expressed the wild-type Stat5 construct (BLG/STAT5) and the constitutively active Stat5 variant (BLG/STAT5ca) exclusively in mammary epithelial cells during pregnancy and lactation. BLG/STAT5 mice exhibited larger alveoli at mid-pregnancy and a delayed onset of involution. Condensed alveoli, a high degree of cellular proliferation, and delayed involution were associated with STAT5ca expression. Elevated levels of beta-casein gene expression were found in BLG/STAT5 and STAT5ca transgenic mice during late pregnancy and lactation, indicating a limiting role for Stat5 under normal physiological conditions. This was accompanied by higher levels of beta-casein secretion into the milk and enhanced growth of pups. Transgenic animals expressing the BLG/STAT5ca transgene were predisposed to tumor formation in the mammary gland. This study extends the functional observations made in cultured mammary epithelial cells and in gene knockout mice. It identifies Stat5 as a multifunctional regulator of mammary cell proliferation, milk protein gene expression, and postlactational apoptosis.

Subcellular knockout of importin ß1 perturbs axonal retrograde signaling.

Subcellular localization of mRNA enables compartmentalized regulation within large cells. Neurons are the longest known cells; however, so far, evidence is lacking for an essential role of endogenous mRNA localization in axons. Localized upregulation of Importin ß1 in lesioned axons coordinates a retrograde injury-signaling complex transported to the neuronal cell body. Here we show that a long 3' untranslated region (3' UTR) directs axonal localization of Importin ß1. Conditional targeting of this 3' UTR region in mice causes subcellular loss of Importin ß1 mRNA and protein in axons, without affecting cell body levels or nuclear functions in sensory neurons. Strikingly, axonal knockout of Importin ß1 attenuates cell body transcriptional responses to nerve injury and delays functional recovery in vivo. Thus, localized translation of Importin ß1 mRNA enables separation of cytoplasmic and nuclear transport functions of importins and is required for efficient retrograde signaling in injured axons.

Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve.

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.

Expression of a carboxy terminally truncated Stat5 with no transactivation domain in the mammary glands of transgenic mice inhibits cell proliferation during pregnancy, delays onset of milk secretion, and induces apoptosis upon involution.

Signal transducer and activator of transcription (Stat5) is a transcription factor, which transduces extracellular cytokine and growth-factor signals to the nuclei of mammalian cells. As a major mediator of prolactin action, it is involved in the regulation of the development, function, and survival of mammary epithelial cells. The carboxyl terminal of Stat5 encodes a transactivation domain (TAD), which interacts with coactivators and is crucial for the transcriptional induction of Stat5 target genes. To study the role of the Stat5 TAD in mediating Stat5 functions, a carboxy terminally truncated Stat5 variant (Stat5Delta750) was directed for expression in the mammary glands of transgenic mice by regulatory sequences of the beta-lactoglobulin (BLG) gene. Expression of Stat5Delta750 in mammary tissue reduced the rates of cell proliferation at mid and late pregnancy. Subsequently, morphological signs of milk secretion upon parturition were delayed. In double-transgenic mice, expression of Stat5Delta750 drastically decreased BLG/luciferase activity during lactation, but did not affect the expression and secretion of the endogenous beta-casein or alpha-lactalbumin into the milk. Expression of Stat5Delta750 also caused an increase in the number of apoptotic cells during mammary involution by a factor of 3 relative to control glands. Our data established a role for the Stat5 TAD in mediating the effects of Stat5 on mammary development, regulation of milk protein gene activity, and cell survival. The full effects of Stat5Delta750 may be partially buffered by the expression of endogenous wild-type Stat5 and the formation of truncated and wild-type heterodimers.

Deregulation of Stat5 expression and activation causes mammary tumors in transgenic mice.

Members of the signal transducers and activators of transcription (Stat) family regulate essential cellular growth and survival functions in normal cells and have also been implicated in tumorigenesis. We have studied the potential role of Stat5 in mammary tumorigenesis by targeting Stat5 variants to the mammary gland of transgenic mice using regulatory sequences of the beta-lactoglobulin gene. Mammary-directed expression of the wild-type Stat5, constitutively activated Stat5 and carboxyl-terminally truncated dominant negative Stat5 forms resulted in mammary tumors with incidence rates of up to 22% and latency periods of 8-12 months. Undifferentiated carcinomas most frequently occurred in mice expressing the carboxyl-terminally truncated Stat5. The more differentiated papillary and micropapillary adenocarcinomas were primarily found in mice overexpressing the native and constitutively active transgenes. Higher levels of translation initiation factor 4E (eIF4E) and cyclin D1 expression but lower levels of activated Stat3 were found in tumors of mice expressing the constitutively active Stat5 when compared to mice expressing the wild-type or truncated forms. A higher expression of the estrogen receptor (ERalpha) was observed in carcinomas compared to other phenotypes. The ability of both forms of Stat5, the transactivating form and the dominant negative form, to participate in oncogenesis indicates that there is more than one mechanism by which Stat5 contributes to this process. The transactivation function of Stat5 is involved in the determination of tumors with a more differentiated phenotype.