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Pyridoxal 5′‐phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ‐aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca\(^{2+}\)‐ and integrin‐binding protein 1 (CIB1) as a PDXP interactor by yeast two‐hybrid screening and find a calmodulin (CaM)‐binding motif that overlaps with the PDXP‐CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca\(^{2+}\) concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP‐CIB1 complex may functionally differ from the PDXP‐Ca\(^{2+}\)‐CaM complex.

Mammalian phosphoglycolate phosphatase (PGP, also known as AUM or glycerol-3-phosphate phosphatase) is a small molecule-directed phosphatase important for metabolite repair and lipid metabolism. Although PGP was first characterized as an enzyme involved in epidermal growth factor (EGF) signaling, PGP protein substrates have remained elusive. Here we show that PGP depletion facilitates fatty acid flux through the intracellular triacylglycerol/fatty acid cycle, and that phosphatidylinositol-4,5-bisphosphate (PIP2), produced in a side branch of this cycle, is critical for the impact of PGP activity on EGF-induced signaling. Loss of endogenous PGP expression amplified both EGF-induced EGF receptor autophosphorylation and Src-dependent tyrosine phosphorylation of phospholipase C-γ1 (PLCγ1). Furthermore, EGF enhanced the formation of circular dorsal ruffles in PGP-depleted cells via Src/PLCγ1/protein kinase C (PKC)-dependent signaling to the cytoskeleton. Inhibition of adipose triglyceride lipase normalized the increased PIP2 content, reduced EGF-dependent PLCγ1 hyperphosphorylation, and decreased the elevated dorsal ruffle formation of PGP-depleted cells. Our data explain how PGP exerts control over EGF-induced cellular protein tyrosine phosphorylation, and reveal an unexpected influence of triacylglycerol turnover on growth factor signaling.

Mammalian phosphatases of the haloacid dehalogenase (HAD) superfamily have emerged as important regulators of physiology and disease. Many of these enzymes are stable homodimers; however, the role of their dimerization is largely unknown. Here, we explore the function of the obligatory homodimerization of chronophin, a mammalian HAD phosphatase known to dephosphorylate pyridoxal 5'-phosphate (PLP) and serine/threonine-phosphorylated proteins. The exchange of two residues in the murine chronophin homodimerization interface (chronophin(A194K,A195K)) yields a constitutive monomer both in vitro and in cells. The catalytic activity of monomeric chronophin toward PLP is strongly impaired. X-ray crystallographic studies of chronophin(A194K,A195K) revealed that dimer formation is essential for an intermolecular arginine-arginine-tryptophan stacking interaction that positions a critical histidine residue in the substrate specificity loop of chronophin for PLP coordination. Analysis of all available crystal structures of HAD hydrolases that are grouped together with chronophin in the C2a-type structural subfamily uncovered a highly conserved mode of dimerization that results in intermolecular contacts involving the substrate specificity loop. Our results explain how the dimerization of HAD hydrolases contributes to their catalytic efficiency and substrate specificity.

The cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate long cytoplasmic extensions, proplatelets, which have the capacity to generate platelets. Although microtubules are the main structural component of proplatelets and microtubule sliding is known to drive proplatelet elongation, the role of actin dynamics in the process of platelet formation has remained elusive. Here, we tailored a mouse model lacking all ADF/n-cofilin–mediated actin dynamics in megakaryocytes to specifically elucidate the role of actin filament turnover in platelet formation. We demonstrate, for the first time, that in vivo actin filament turnover plays a critical role in the late stages of platelet formation from megakaryocytes and the proper sizing of platelets in the periphery. Our results provide the genetic proof that platelet production from megakaryocytes strictly requires dynamic changes in the actin cytoskeleton.

Class I B phosphoinositide 3-kinase γ (PI3Kγ) elicits various immunologic and cardiovascular responses; however, the molecular basis for this signal heterogeneity is unclear. PI3Kγ consists of a catalytic p110γ and a regulatory p87 PIKAP (p87, also p84) or p101 subunit. Hitherto p87 and p101 are generally assumed to exhibit redundant functions in receptor-induced and G protein βγ (Gβγ)-mediated PI3Kγ regulation. Here we investigated the molecular mechanism for receptor-dependent p87/p110γ activation. By analyzing GFP-tagged proteins expressed in HEK293 cells, PI3Kγ-complemented bone marrow–derived mast cells (BMMCs) from p110γ -/- mice, and purified recombinant proteins reconstituted to lipid vesicles, we elucidated a novel pathway of p87-dependent, G protein–coupled receptor (GPCR)-induced PI3Kγ activation. Although p101 strongly interacted with Gβγ, thereby mediating PI3Kγ membrane recruitment and stimulation, p87 exhibited only a weak interaction, resulting in modest kinase activation and lack of membrane recruitment. Surprisingly, Ras-GTP substituted the missing Gβγ-dependent membrane recruitment of p87/p110γ by direct interaction with p110γ, suggesting the indispensability of Ras for activation of p87/p110γ. Consequently, interference with Ras signaling indeed selectively blocked p87/p110γ, but not p101/p110γ, kinase activity in HEK293 and BMMC cells, revealing an important crosstalk between monomeric and trimeric G proteins for p87/p110γ activation. Our data display distinct signaling requirements of p87 and p101, conferring signaling specificity to PI3Kγ that could open up new possibilities for therapeutic intervention.

Abstract —There is evidence that telomeres, the ends of chromosomes, serve as clocks that pace cellular aging in vitro and in vivo. In industrialized nations, pulse pressure rises with age, and it might serve as a phenotype of biological aging of the vasculature. We therefore conducted a twin study to investigate the relation between telomere length in white blood cells and pulse pressure while simultaneously assessing the role of genetic factors in determining telomere length. We measured by Southern blot analysis the mean length of the terminal restriction fragments (TRF) in white blood cells of 49 twin pairs from the Danish Twin Register and assessed the relations of blood pressure parameters with TRF. TRF length showed an inverse relation with pulse pressure. Both TRF length and pulse pressure were highly familial. We conclude that telomere length, which is under genetic control, might play a role in mechanisms that regulate pulse pressure, including vascular aging.

Telomerase activity is detected in many immortalized cell lines. Recent studies suggest that terminal differentiation of some of these cell lines is associated with a reduction in telomerase activity. However, the question remains whether the reduction in telomerase activity results from terminal differentiation or from cessation of cellular proliferation. This was explored in the megakaryocytic cell line CHRF-288-11. Cells were treated with phorbol 12-myristate 13-acetate (PMA), which induces terminal differentiation of CHRF-288-11 cells, EGTA, serum depletion, and okadaic acid. All treatments resulted in cessation of proliferation. Except for okadaic acid, these treatments also induced inhibition of telomerase within 7 days. Restoring the original growth conditions of cells treated with PMA, EGTA and serum depletion resulted in the reversal of telomerase inhibition and an acceleration of proliferation. Apparent inhibition of telomerase was observed to follow the cessation of proliferation, whereas enhanced telomerase activity was noted to precede acceleration in proliferation. Thus, telomerase activity usually reflects the proliferative status rather than the differentiated status of CHRF-288-11 cells.