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CaM kinase IV regulates lineage commitment and survival of erythroid progenitors in a non-cell-autonomous manner.

Wayman GA, Walters MJ, Kolibaba K, Soderling TR, Christian JL - J. Cell Biol. (2000)

Bottom Line: Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis.These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage.Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.

View Article: PubMed Central - PubMed

Affiliation: Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA.

ABSTRACT
Developmental functions of calmodulin-dependent protein kinase IV (CaM KIV) have not been previously investigated. Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis. Xenopus embryos in which CaM KIV activity is either upregulated or inhibited show that hematopoietic precursors are properly specified, but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of CaM KIV activity causes commitment of hematopoietic precursors to myeloid differentiation at the expense of erythroid differentiation, on the other hand, constitutive activation of CaM KIV induces erythroid precursors to undergo apoptotic cell death. These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage. Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.

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Enzymatically active CaM KK is present in Xenopus embryos. Whole Xenopus embryos, at the indicated developmental stages, and dissected dorsal and ventral halves of early gastrulae were homogenized and four embryo equivalents were assayed for induction of Ca2+–CaM-independent activation of recombinant CaM KIV (graph) or were subjected to Western blot analysis (insets). The control lane on the Western blot contains 50 ng of purified rat CaM KKα. Relative CaM KK activity is presented as the mean fold ± SD stimulation of CaM KIV activity as determined in triplicate assays.
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Figure 4: Enzymatically active CaM KK is present in Xenopus embryos. Whole Xenopus embryos, at the indicated developmental stages, and dissected dorsal and ventral halves of early gastrulae were homogenized and four embryo equivalents were assayed for induction of Ca2+–CaM-independent activation of recombinant CaM KIV (graph) or were subjected to Western blot analysis (insets). The control lane on the Western blot contains 50 ng of purified rat CaM KKα. Relative CaM KK activity is presented as the mean fold ± SD stimulation of CaM KIV activity as determined in triplicate assays.

Mentions: CaM KK, the upstream activator of CaM KIV, is also expressed embryonically. As shown in Fig. 4, the temporal and spatial pattern of expression of embryonic CaM KK mirrors its activity profile. Antisera directed against rat CaM KK detected an immunoreactive band of ∼68 kD that comigrated with recombinant rat CaM KKα on Western blots of Xenopus egg and embryo extracts. This protein increased in abundance by tadpole stage 42, at which time a second immunoreactive protein of ∼70 kD was also detected. The size of this higher molecular weight species is consistent with it being the Xenopus ortholog of CaM KKβ. The increase in CaM KK protein levels at the tadpole stage correlated with an approximately threefold increase in CaM KK activity. To determine whether CaM KK activity is spatially restricted, we analyzed protein expression and activity in extracts isolated from dissected dorsal or ventral halves of early (stage 10) gastrulae. CaM KK protein and activity were detected in dorsal and ventral halves of embryos.


CaM kinase IV regulates lineage commitment and survival of erythroid progenitors in a non-cell-autonomous manner.

Wayman GA, Walters MJ, Kolibaba K, Soderling TR, Christian JL - J. Cell Biol. (2000)

Enzymatically active CaM KK is present in Xenopus embryos. Whole Xenopus embryos, at the indicated developmental stages, and dissected dorsal and ventral halves of early gastrulae were homogenized and four embryo equivalents were assayed for induction of Ca2+–CaM-independent activation of recombinant CaM KIV (graph) or were subjected to Western blot analysis (insets). The control lane on the Western blot contains 50 ng of purified rat CaM KKα. Relative CaM KK activity is presented as the mean fold ± SD stimulation of CaM KIV activity as determined in triplicate assays.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169444&req=5

Figure 4: Enzymatically active CaM KK is present in Xenopus embryos. Whole Xenopus embryos, at the indicated developmental stages, and dissected dorsal and ventral halves of early gastrulae were homogenized and four embryo equivalents were assayed for induction of Ca2+–CaM-independent activation of recombinant CaM KIV (graph) or were subjected to Western blot analysis (insets). The control lane on the Western blot contains 50 ng of purified rat CaM KKα. Relative CaM KK activity is presented as the mean fold ± SD stimulation of CaM KIV activity as determined in triplicate assays.
Mentions: CaM KK, the upstream activator of CaM KIV, is also expressed embryonically. As shown in Fig. 4, the temporal and spatial pattern of expression of embryonic CaM KK mirrors its activity profile. Antisera directed against rat CaM KK detected an immunoreactive band of ∼68 kD that comigrated with recombinant rat CaM KKα on Western blots of Xenopus egg and embryo extracts. This protein increased in abundance by tadpole stage 42, at which time a second immunoreactive protein of ∼70 kD was also detected. The size of this higher molecular weight species is consistent with it being the Xenopus ortholog of CaM KKβ. The increase in CaM KK protein levels at the tadpole stage correlated with an approximately threefold increase in CaM KK activity. To determine whether CaM KK activity is spatially restricted, we analyzed protein expression and activity in extracts isolated from dissected dorsal or ventral halves of early (stage 10) gastrulae. CaM KK protein and activity were detected in dorsal and ventral halves of embryos.

Bottom Line: Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis.These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage.Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.

View Article: PubMed Central - PubMed

Affiliation: Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA.

ABSTRACT
Developmental functions of calmodulin-dependent protein kinase IV (CaM KIV) have not been previously investigated. Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis. Xenopus embryos in which CaM KIV activity is either upregulated or inhibited show that hematopoietic precursors are properly specified, but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of CaM KIV activity causes commitment of hematopoietic precursors to myeloid differentiation at the expense of erythroid differentiation, on the other hand, constitutive activation of CaM KIV induces erythroid precursors to undergo apoptotic cell death. These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage. Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.

Show MeSH
Related in: MedlinePlus