Structure of the human protein kinase MPSK1 reveals an atypical activation loop architecture.

The activation segment of protein kinases is structurally highly conserved and central to regulation of kinase activation. Here we report an atypical activation segment architecture in human MPSK1 comprising a beta sheet and a large alpha-helical insertion. Sequence comparisons suggested that similar activation segments exist in all members of the MPSK1 family and in MAST kinases. The consequence of this nonclassical activation segment on substrate recognition was studied using peptide library screens that revealed a preferred substrate sequence of X-X-P/V/I-phi-H/Y-T*-N/G-X-X-X (phi is an aliphatic residue). In addition, we identified the GTPase DRG1 as an MPSK1 interaction partner and specific substrate. The interaction domain in DRG1 was mapped to the N terminus, leading to recruitment and phosphorylation at Thr100 within the GTPase domain. The presented data reveal an atypical kinase structural motif and suggest a role of MPSK1 regulating DRG1, a GTPase involved in regulation of cellular growth.

Prenatal exposure to cocaine decreases adenylyl cyclase activity in embryonic mouse striatum.

Adenylyl cyclase activity was measured in the striatum of naive mice as a function of age and in mice exposed in utero to cocaine. In naive Swiss-Webster mice, basal and forskolin-stimulated adenylyl cyclase activity increased gradually from embryonic day 13 (E13) until 2-3 weeks of age when activity peaked before decreasing slightly to adult levels. The ability of the dopamine D1 receptor agonist, SKF 82958, to stimulate adenylyl cyclase activity also increased in magnitude until P15. In a separate study, pregnant Swiss-Webster mice were injected twice daily with cocaine (15 mg/kg, s.c.) or an equal volume of saline from E10 to E17. Adenylyl cyclase activity was measured in the striatum of E18 embryos. Basal adenylyl cyclase activity was significantly reduced following prenatal exposure to cocaine. Likewise, the ability of forskolin or SKF 82958 to stimulate adenylyl cyclase was attenuated following cocaine exposure. DeltaFosB was not induced, contrary to what is seen in adult mice. These results demonstrate a functional change in a critical signal transduction pathway following chronic in utero exposure to cocaine that might have profound effects of the development of the brain. Alterations in the cAMP system may underlie some of the deficits seen in humans exposed in utero to cocaine.

Nucleocytoplasmic shuttling of STK16 (PKL12), a Golgi-resident serine/threonine kinase involved in VEGF expression regulation.

PKL12/STK16 protein is the first identified mammalian member of a ser/thr kinase subfamily that is conserved across several kingdoms, with a broad expression pattern in murine tissues and cell types. Endogenous STK16 subcellular localization was evaluated by indirect immunofluorescence in NIH/3T3 and NRK cells, demonstrating a Golgi-associated pattern that appears to be independent of signals provided by integrin pathways. When cells were treated with brefeldin A (BFA) or nocodazole, drugs that promote Golgi disorganization, we observed STK16 translocation to the nuclear compartment. Constitutive overexpression of this protein by retroviral vectors also promotes accumulation of STK16 in the nuclear compartment, as shown by subfractionation studies. A kinase-dead STK16 mutant (E202A) was used to demonstrate that both the Golgi association and the nuclear translocation capabilities seem to be independent of the STK16 kinase activity. In addition, we show that STK16 overexpression in several cell lines enhances their capacity to produce and secrete VEGF. To confirm these data in vivo, we injected tumor cells overexpressing STK16 into immunodeficient BALBc/SCID mice. HT1080-derived tumors overexpressing STK16 showed increased volume and number of blood vessels compared to controls. Altogether, these data concur with previous reports suggesting a potential role for STK16 as a transcriptional co-activator.

17beta-Estradiol promotes striatal medium size spiny neuronal maturation in vitro.

Gender differences exist in the development of the nigrostriatal dopamine system, and in the incidence and course of pediatric and adult neuropsychiatric diseases in which this system is implicated. The medium size spiny neuron (MSN) is the major output neuron of the caudate nucleus. It receives a large dopaminergic input from the substantia nigra, and 96% of the MSNs express DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein and key mediator of dopamine function. There are few examples, however, of direct effects of sex hormones, including 17beta-estradiol (E(2)), on the MSN. We report that in vitro, E(2) (10-50 nM) promotes MSN phenotypic maturation, as determined by increased soma size, neurite length, and DARPP-32 protein levels. Treatment with the 'anti-estrogen' ICI 182,780 or the partial-agonist tamoxifen also increases DARPP-32 levels, but when added to E(2), ICI 182,780 only prevents the increase in DARPP-32 levels and increase in soma size and neurite length. Surprisingly, maturation effects are more robust in cells derived exclusively from female embryos. Western blot analysis of protein lysates and immunocytochemistry of cultured MSNs reveals the presence of the estrogen receptor beta (ERbeta). These data suggest that ERbeta may mediate the differentiating effect of E(2) on embryonic MSNs, and provide new avenues of investigation for the role of sex hormones in the development of the striatum and in diseases affecting the basal ganglia.

Functional interaction between the Ser/Thr kinase PKL12 and N-acetylglucosamine kinase, a prominent enzyme implicated in the salvage pathway for GlcNAc recycling.

PKL12 (STK16) is a ubiquitously expressed Ser/Thr kinase, not structurally related to the well known subfamilies, with a putative role in cell adhesion control. Yeast two-hybrid protein interaction screening was used to search for proteins that associate with PKL12 and to delineate signaling pathways and/or regulatory circuits in which this kinase participates. One positive clone contained an open reading frame highly similar to N-acetylglucosamine kinase (GlcNAcK) of several species. The PKL12/GlcNAcK interaction was further confirmed both in vitro and in vivo. Protein expression analysis of GlcNAcK using a specific rabbit antiserum displayed a ubiquitous pattern in cell lines and animal tissues. Subcellular localization studies showed that GlcNAcK is a cytoplasmic protein with a dual subcellular localization, distributed between the perinuclear and peripheral cell reservoirs. After overexpression, GlcNAcK localizes in vesicular structures associated mainly with the cell membrane and colocalizes with the PKL12 protein. GlcNAcK is not otherwise a substrate for PKL12 activity and PKL12 does not appear to influence GlcNAcK activity either in vitro or in vivo. In vitro kinase assays have nonetheless revealed that functional GlcNAcK, although not able to modulate autophosphorylation of PKL12, greatly influences PKL12 kinase activity on a defined substrate protein. These results are interpreted to indicate a potential in vivo role for GlcNAcK in PKL12 translocation and a tentative regulatory role for PKL12-mediated phosphorylation on substrate proteins.