Human HTm4 is a hematopoietic cell cycle regulator
José L. Donato, Jon Ko, Jeffery L. Kutok, Tao Cheng, Taro Shirakawa, Xiao-Quan Mao, David Beach, David T. Scadden, Mohamed H. Sayegh, Chaker N. Adra
José L. Donato, Jon Ko, Jeffery L. Kutok, Tao Cheng, Taro Shirakawa, Xiao-Quan Mao, David Beach, David T. Scadden, Mohamed H. Sayegh, Chaker N. Adra
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Article

Human HTm4 is a hematopoietic cell cycle regulator

Abstract

Proper control of cell cycle progression is critical for the constant self-renewal, differentiation, and homeostasis of the hematopoietic system. Cells of all types share the common cell cycle regulators. The different expression patterns of common regulators, in a broad sense, define cell-type or lineage specificity. However, there remains the possibility of hematopoietic cell cycle regulators tailored to the demands of the hematopoietic system. Here we describe a novel protein, HTm4, which serves as a hematopoietic cell cycle regulator. Our data indicate that HTm4 is expressed in hematopoietic tissues and is tightly regulated during the differentiation of hematopoietic stem cells. It binds to cyclin-dependent kinase–associated (CDK-associated) phosphatase-CDK2 (KAP-CDK2) complexes, and the three proteins demonstrate similar patterns of cellular expression in human lymphoid tissues. HTm4 stimulates the phosphatase activity of KAP, and its C-terminal region is required for binding to KAP-CDK2 complexes and the modulation of KAP activity. Overexpression of HTm4 can cause cell cycle arrest at the G0/G1 phase. Thus, HTm4 is a novel hematopoietic modulator for the G1-S cell cycle transition.

Authors

José L. Donato, Jon Ko, Jeffery L. Kutok, Tao Cheng, Taro Shirakawa, Xiao-Quan Mao, David Beach, David T. Scadden, Mohamed H. Sayegh, Chaker N. Adra

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(a) Binding of HTm4 to KAP in yeast two-hybrid assay. Constructs listed ...
(a) Binding of HTm4 to KAP in yeast two-hybrid assay. Constructs listed in the upper and lower rows were in pGADGH, whereas those to the left of the first column were in pVJLII. HTm4 listed here represents the CHTm4. MEK, byr, and lamin are negative controls. The positive results are shown as colony growth on selective medium and blue color reaction in the presence of β-gal. (b) Isolation of KAP from U937 and KU812 lysates using GST-CHTm4 fusion protein. GST-C represents GST-CHTm4. Lysates derived from U937 and KU812 are absorbed with either GST-CHTm4 or GST-coupled beads, and bound proteins were analyzed in a Western blot assay for the presence of KAP. (c) Coimmunoprecipitation of HA-tagged KAP and HTm4. CTL, pCMV control vector. The source of the samples is listed on the top. The first two columns on the left are lysates alone and the remaining two are after immunoprecipitation with anti-HA. Samples are analyzed by the Western blot technique using anti-HTm4 for the top two panels and anti-HA for the bottom two. The identified proteins are listed on the right. (d) HTm4 and KAP form a physiological complex. The source of the samples is denoted on the top; the first column from the left is U937 lysate alone; the second and third are after immunoprecipitation with anti-KAP and IgG control, respectively. The presence of HTm4 is indicated on the right after Western blot analysis. Representative figures of at least five experiments.