A breakdown in the regulation of adipocyte lipid storage and mobilization pathways can contribute to increased levels of NEFA in the circulation, which is an established risk factor for the development of insulin resistance in type II diabetes and related disorders Gesta et al , ; Guilherme et al , Phosphorylation of HSL occurs on multiple sites, including Ser, which stimulates catalytic activity and Ser, which is believed to be mutually exclusive with phosphorylation of HSL at the non-PKA site Ser Anthonsen et al , ; Watt et al , Thus, hormonal cues that signal systemic energy induce HSL phosphorylation at Ser by PKA, which contributes to adipocyte lipolysis to maintain whole-body energy homeostasis.
Prominent among these is the tumour suppressor kinase LKB1 Sakamoto et al , ; Shaw et al , In the context of adipocytes, evidence suggests that AMPK is activated as a consequence of constitutively ongoing reesterification that consumes energy Gauthier et al , Thus, stimulation of adipocyte lipolysis through PKA activation triggers, in turn, a negative feedback mechanism involving AMPK to match the rate of lipolysis to energy supply.
The mechanism s underlying coordination of these apparently opposing activities under conditions of acute systemic metabolic needs remain elusive. Stable complex formation of these kinases was, however, not affected by treatment of cells with either isoproterenol or ionomycin Supplementary Figure S1C.
Protein lysates were prepared and probed with the indicated antibodies. FSK was either present ab initio lane 8 , added during the last 5, 10, 20, 30 or 45 min of starvation lanes 3—8 or omitted lane 2. Equal amounts of protein lysates were subjected to immunoblotting with the indicated antibodies.
Proteins were subjected to immunoblotting with the indicated antibodies. NEFA were measured in the incubation medium. Bars represent the mean NEFA release from three independent experiments. Immunoblots are representative of at least three independent experiments. Further verification of the phosphosites was attempted by solid-phase sequencing of the radiolabelled peptides. After each cycle of N-terminal Edman degradation, liberated single amino acids were collected and spotted onto DEAE cellulose.
The respective phosphorylated residues were then detected by autoradiography. Dha, dehydroala. However, the anti-phospho Ser antibodies failed now to recognize the phospho-Ser epitope Figure 3A , lane 4. Signals obtained by autoradiography indicate phosphorylation at Ser lanes 3 and 4. In this double-phosphorylated form, phosphorylation of Thr but not Ser was recognized by the corresponding antibodies.
From blotting with anti-pSer or anti-pSer antibodies, but also from phosphopeptide mapping performed previously by us Henriksson et al. To identify SIK2-interacting proteins, we employed both focused and more general unbiased strategies. HA—SIK2 was then immunoisolated using anti-HA—agarose, and co-immunoprecipitating proteins were identified by western blotting. Lysates from GFP-expressing cells were used as a negative control.
As shown previously Henriksson et al. This suggests that phosphorylation of Ser is important for cAMP-dependent regulation of the interaction of SIK2 with some of its molecular targets.
When identifying co-immunoprecipitated proteins by mass fingerprinting, we noted a large number of peptides originating from the regulatory as well as the catalytic subunit of PP2A supplementary material Fig.
Immunoprecipitates of both wild-type and SerAla SIK2 contained PP2A and, in this setting using a non-quantitative form of mass spectrometry , we did not observe any marked differences between samples originating from cells stimulated with or without forskolin. Considering the important role of GLUT4 in glucose uptake, we next investigated the impact of SIK2 on basal and insulin-stimulated glucose uptake in primary rat adipocytes by monitoring the uptake of 14 C glucose in cells expressing GFP or the wild-type or kinase-inactive ThrAla SIK2.
Glucose uptake in the presence of insulin was not affected by the expression of wild-type SIK2. Silencing of the respective proteins was confirmed by western blotting supplementary material Fig. Similar results were obtained when analyzing lysates from adipocytes isolated and pooled from 11—week-old mice seven per genotype, data not shown. We thereafter analysed glucose uptake in the absence and presence of insulin.
As shown in Fig. The fold-increase in glucose uptake in response to insulin stimulation was, however, not different in inhibitor-treated cells Fig.
Pre-treatment with MRT at slightly higher concentrations than that of HG resulted in a similar inhibition of basal glucose uptake Fig. These data collectively suggest that SIK2 activity is involved in maintaining basal glucose uptake in primary rat adipocytes. Our data from rodent adipocytes make SIK2 an interesting protein to study in relation to human adipose tissue function and dysfunction.
To do so, we used human adipocytes isolated from surgical adipose tissue biopsies. Regulation of SIK2 signalling in human adipocytes. Representative immunoblots are shown. So far very few studies have attempted to identify molecular targets or biological roles of SIK2 in adipocytes — even though SIK2 levels are several-fold higher in adipose tissue than in any other tissue.
This highlights a potential role for SIK2 in human adipose tissue function and in the development of obesity and associated disorders.
To monitor site-specific phosphorylation of CRTC2 and CRTC3, we employed phosphospecific antibodies against Ser and Ser, respectively, both of which are sites previously suggested to be important for the localisation of these proteins Clark et al. Owing to the lack of commercially available tools of sufficient quality, we were not able to confidently study the effect of SIK2 on these sites in adipocytes. We attempted to directly map the sites phosphorylated in CRTC2 when coexpressed with wild-type SIK2 in primary adipocytes, but this analysis was hampered by the poor yield of CRTC2 and a tryptic cleavage pattern generating several peptides that were too large for detection.
Existing customer? Sign in. The secret to supporting increasing healthy populations of wildlife is establishing complete habitat. Habitat that supplies all the life sustaining needs for all ages, for all seasons, and against all threats.
Soil Moisture: Dry- Medium. Regions: 1, 2, 3, 4, 5, 6. Soil Moisture: All Soils. Soil Moisture: Wet Site. Soil Moisture: Dry Site. Soil Moisture: Dry and Wet Site. We're here to help We're Native Seed Experts, with over 20 years of growing and supplying clean, high quality, regionally adapted Native Seed. John Seymour - President. While our in vitro, ex vivo and in vivo observations were all consistent with a positive for of AKAP12 in the endothelial cell migration, and with effects observed in zebrafish, 12 previous studies using cultured cells reached opposite conclusions.
However, consistent with data from cancer cells where AKAP12 was reported to inhibit motility and cancer cell invasiveness, 41 early reports of AKAP12 in endothelial cells linked it with the inhibition of angiogenesis via effects linked to HDAC7 42 and matrix metalloproteinase 9.
This was reinforced by a study published during the preparation of this manuscript that clearly linked VASP with adhesion filopodia formation in osteosarcoma cells. Given that AKAP12 was apparently required to promote endothelial cell migration and angiogenesis in vitro and in vivo, we looked at its ability to interact with other proteins involved in regulating cell motility.
Littermates were used throughout this study. Umbilical cords were obtained from a local hospital and umbilical vein endothelial cells were isolated and cultured as described, 56 and used only up to passage 1.
The use of human material in this study conforms to the principles outlined in the Declaration of Helsinki 57 and the isolation of endothelial cells was approved in written form by the ethic committee of the Goethe University. Murine lung endothelial cells were isolated and cultured as described. Percentage of wound closure was analysed using the IncuCyte A software. Spheroids containing cells were generated as described, 63 in endothelial basal medium EBM supplemented with 2.
After 24 hours in a collagen gel, angiogenesis was quantified by measuring the cumulative length of all the capillary like sprouts originating from an individual spheroid using a computer assisted microscope Zeiss, Oberkochen, Germany and ImageJ.
Then retinas were washed three times in Pblec buffer 0. For secondary detection, Alexa Fluor—coupled secondary antibodies were used. Cells were allowed to migrate for 13 hours before cells were fixed, stained and imaged as detailed above. The whole procedure was repeated one week later with three additional, independent batches of endothelial cells, resulting in 6 AKAP12 and 6 control immunoprecipitations in total.
Five micrograms of purified bovine serum albumin coupled to Affigel 15 were used as control. Lysates were cleared by centrifugation, incubated with the corresponding affinity matrix for 1. Thiols were alkylated with 10 mM chloroacetamid. Digestion was stopped by adding trifluoroacetic acid to a final concentration of 0. Purification and elution of peptides was performed as described in.
Fullscan data were acquired in profile and fragments in centroid mode by Xcalibur software. For data analysis MaxQuant v1. Missing LFQ values were replaced by random background values.
Significant interacting proteins were determined by Students t test. PMB and IF wrote the paper. All authors read and approved final draft of the manuscript. Acta Physiol. L and I. National Center for Biotechnology Information , U.
Acta Physiologica Oxford, England. Expression of tyrosine kinases FAK and Pyk2 in human astrocytomas. Acta Neuropathol. Hecker, T. Cancer Res. Ding, L. Expression of focal adhesion kinase and phosphorylated focal adhesion kinase in human gliomas is associated with unfavorable overall survival. Wu, Y. Liu, M. The effect of epidermal growth factor receptor variant III on glioma cell migration by stimulating ERK phosphorylation through the focal adhesion kinase signaling pathway.
Natarajan, M. FAK signaling in anaplastic astrocytoma and glioblastoma tumors. Cancer J. Jones, G. Loss of focal adhesion kinase FAK inhibits epidermal growth factor receptor-dependent migration and induces aggregation of nh 2 -terminal FAK in the nuclei of apoptotic glioblastoma cells.
Liu, T. Inhibition of both focal adhesion kinase and insulin-like growth factor-I receptor kinase suppresses glioma proliferation in vitro and in vivo. Cancer Ther. Seufert, S. PPAR gamma activators: off-target against glioma cell migration and brain invasion.
PPAR Res. Zheng, Q. Neuro-Oncology 16 , — Devroe, E. Hergovich, A. MOB control: reviewing a conserved family of kinase regulators. Bothos, J. Human LATS1 is a mitotic exit network kinase. Kohler, R. Gundogdu, R. MOB Mps one Binder proteins in the hippo pathway and cancer. Gomez, V. Cerami, E. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. Lin, C. The promotion of neurite formation in Neuro2A cells by mouse Mob2 protein.
FEBS Lett. Chen, L. Lou, L. Swaney, J.Feb 01, · Role of PKA-mediated phosphorylation of SIK2 for the regulation of CRTCs and HDAC4 by cAMP in adipocytes. SIK2 is regulated by cAMP–PKA signalling in adipocytes through phosphorylation at four sites – Ser, Ser, Thr and Ser – and we have shown previously that this is associated with the binding of SIK2 to proteins (Henriksson et al., ).