A Dissertation Entitled JAK2 Tyrosine Kinase Phosphorylates and Is

A Dissertation

Entitled

JAK2 Tyrosine Kinase Phosphorylates and is Negatively Regulated by the Centrosomal

Protein Ninein

Jennifer Jay

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in Biology

______Dr. Maria Diakonova, Committee Chair

______Dr. Patricia R. Komuniecki, Dean College of Graduate Studies

The University of Toledo

May 2015

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

JAK2 Tyrosine Kinase Phosphorylates and is Negatively Regulated by the Centrosomal Protein Ninein

Jennifer Jay

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology

The University of Toledo May 2015

Janus Kinase 2 (JAK2) is a non-receptor tyrosine kinase that is activated by two- thirds of the cytokine receptor superfamily including receptors to interferon-γ, growth hormone and prolactin. Upon ligand binding to its receptor, JAK2 becomes activated and can phosphorylate downstream targets that lead to diverse physiological responses. Even though JAK2 plays an important role in cytokine signaling, its subcellular localization is still under debate. We show that both inactive and active JAK2 (pJAK2) localizes around the mother centrioles where it partially colocalizes with ninein, a protein involved in microtubule (MT) nucleation and anchoring. We demonstrate that depletion of JAK2 or using JAK2-null cells, results in microtubule anchorage defects at the mother centriole and an increased number of cells with mitotic defects; however, MT nucleation is unaffected. Additionally, loss of JAK2 leads to an increase in separated centrosomes in interphase cells. We show that JAK2 directly phosphorylates the N-terminus of ninein while the C-terminus of ninein inhibits JAK2 kinase activity in vitro. Overexpression of either C-terminal or WT ninein decreases JAK2 activation in cells, causing a decrease in prolactin and interferon-γ induced tyrosyl phosphorylation of STAT1 and STAT5, iii

downstream targets of JAK2. Down-regulation of endogenous ninein increased JAK2 activation and subsequently STAT1 and STAT5 tyrosyl phosphorylation. These results indicate that JAK2 is a novel member of the centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways.

Acknowledgements

I would like to thank my advisor, Dr. Maria Diakonova, for allowing me to complete my Ph.D. under her supervision. Through her guidance and support I have thrived and I would not be the scientist I am today without her push. I would also like to thank my committee members for their support. I would like to thank all of the previous and current lab members, without them I would not be here today. Their support and friendship kept me going during some hard times. I would also like to thank previous and former members of the Biology Department at UT. I have made so many friends and have felt so at home here from the beginning. UT truly feels like a second home to me. I would like to also thank my family: my parents, Gary and Linda Jay, my brother and sister-in-law, Jeremy and Katie Jay, my niece, Samantha Jay, my in-laws, Barb and Bud, brother-in-laws, Andy and Matt, and sister-in-law, Tammy. Without the support of my entire family I would not be the person I am today, and I am truly grateful to have all of them in my life. Last but not least I want to thank my fiancée, Lori. Without her love, support, and faith in me, I would not be where I am today. I am so grateful for her patience, encouragement, and humor over these last few years, and her willingness to stay with me. She makes me a better person and every day I am thankful that I found her.

Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Table of Contents ...... vi

List of Figures ...... vii

List of Abbreviations ...... ix

I. Introduction…………………… ...... 1

II. Material and Methods...... 15

III. Results……………………………………………………………..…………………26

IV. Discussion………………...... 61

References ...... 68

List of Figures

Figure 1. JAK2 Structure...... 1

Figure 2. Centrosome Structure ...... 6

Figure 3. Centrosome Duplication...... 9

Figure 4. Ninein Structure ...... 11

Figure 5. Ninein Localization During the Cell Cycle…………………………………....13

Figure 6. Active JAK2 tyrosine kinase localizes to the centrosome ...... 27-28

Figure 7. Synchronized HeLa Cells ...... 30

Figure 8. Localization of JAK2 During the Cell Cycle ...... 31-32,34

Figure 9. JAK2 Regulates Aster Formation and Microtubule Release ...... 37-38,40

Figure 10. Endogenous JAK2 Associates with Endogenous Ninein and WT and

C-ninein ...... 41-42

Figure 11. Endogenous JAK2 copurified with Endogenous Ninein in the Same

Centrosomal Fractions ...... 44

Figure 12. Recombinant JAK2 Tyrosyl Phosphorylates the N-terminus of Ninein While

C-terminus of Ninein Inactivates JAK2 activity in vitro ...... …..45-48

Figure 13. C-terminal Ninein Decreases JAK2 Tyrosyl Kinase Activity in vivo ...... 50,52

Figure 14. WT and C-terminal Ninein Decrease Endogenous JAK2 Autophosphorylation

and Subsequently STAT1 and STAT5 Tyrosyl Phosphorylation ...... 53-54,56

Figure 15. Loss of JAK2 Leads to Mitotic Errors ...... 57

Figure 16. Loss of JAK2 causes centrosome splitting ...... 60

Figure 17. Model of JAK2 and Ninein Interaction at the Centrosome...... 63

Figure 18. The Centrosome as the Center of Signal Transduction in the Cell...... 66 vii

List of Abbreviations

ER ...... Endoplasmic Reticulum

FACS...... Fluorescence-activated Cell Sorting FERM ...... band 4.1, erzin, radixin and moesin

GFP ...... Green Fluorescent Protein GSK3β ...... Glycogen Synthase Kinase-3β GST ...... Glutathione S-transferase hNinein ...... Human Ninein

IFNγ ...... γ-interferon

JAK2 ...... Janus Kinase 2 JH ...... JAK Homology Domain

MAPK ...... Mitogen-Activated Protein Kinase MT...... Microtubules MTOC ...... Microtubule Organizing Center

Nek2 ...... NimA-related protein kinase 2

PBS ...... Phosphate Buffer Saline PCM ...... Pericentriolar Material PI3K ...... Phosphatidylinositol 3-Kinase pJAK2 ...... Autophosphorylated JAK2 PKA...... Protein Kinase A PRL ...... Prolactin PTP-BL ...... Protein Tyrosine Phosphatase-Basophil-like PY ...... Phosphotyrosine

SH2 ...... Src-homology-2 SOCS...... Suppressor of Cytokine Signaling STAT...... Signal Transducers and Activators of Transcription

V617F JAK2 ...... Constitutively Active JAK2 VEGF ...... Vascular Endothelial Growth Factor

WT ...... Wild-type

γ-TURC ...... γ-Tubulin Ring Complex

viii

Chapter One

Introduction

JAK

Janus kinase 2 (JAK2) is a non-receptor tyrosine kinase that belongs to the Janus family of cytoplasmic tyrosine kinases that also includes JAK1, JAK3 and TYK2. JAK2 is ubiquitously expressed and contains seven JAK homology domains (JH1-JH7, see Figure

1). At the C-terminus is the kinase domain (JH1) which contains an activation

Figure 1. JAK2 Structure. JAK2 contains seven JAK2 homology (JH) domains. At the N-terminus, JH4-7 makes up the FERM domain, which is responsible for JAK2-receptor interaction. The FERM domain can be phosphorylated at Y119 and Y221 which reduces JAK2 kinase activity. Next is the SH2-like JH3 domain and its function is still unknown. JH2 and JH1 are the pseudokinase and kinase domains, respectively. The pseudokinase domain interacts with the kinase domain, inhibiting JAK2 kinase activity (Saharinen et al., 2000). Recently, it was found that the pseudokinase domain can phosphorylate S523 and Y570, reducing JAK2 kinase activity. The kinase domain contains an activation loop and auto- and/or trans-phosphorylation of Y1007/Y1008 is required for JAK2 kinase activation (modified from Vainchenker and Constantinescu, 2013).

loop. Phosphorylation of Tyr1007/1008 in the activation loop leads to JAK2 kinase 1

activation. The pseudokinase domain (JH2) interacts with the kinase domain to inhibit

JAK2 activity (Saharinen et al., 2000). Mutations and deletion of the pseudokinase domain enhances JAK2 kinase activity (Chen et al., 2000; Lindauer and Loerting, 2001; Saharinen et al., 2002). Until recently, the pseudokinase domain was thought to have no true kinase activity; however, the pseudokinase domain can phosphorylate Ser523 and Tyr570 of JAK2 resulting in a reduction of JAK2 kinase activity (Feener, 2004; Ishida-Takahashi, 2006;

Mazurkiewicz-Munoz, 2006; Ungureanu, 2011). The Src-homology-2 (SH2)-like JH3 domain, is homologous to the SH2 domain; however it is unable to bind and interact with phosphotyrosines and therefore its function is unknown (Harpur, 1992; Wilks et al., 1991;

Kampa and Burnside, 2000). At the N-terminus JH4-7 contain the FERM (band 4.1, erzin, radixin and moesin) domain which is responsible for JAK2 interaction with the cytokine receptors by binding to the Box 1 region of the receptor. The FERM domain can interact with the JH1 domain, regulating JAK2 kinase activity (Argetsinger et al., 2004).

Phosphorylation of Tyr119 and Tyr221 in the FERM negatively regulates kinase activation

(Argetsinger et al., 2004). Mutations or deletions in the FERM domain result in increased kinase activity (Zhao et al., 2010).

JAK2 is responsible for transmission of signals from two thirds of the cytokine receptor superfamily including prolactin, γ-interferon, interleukins, growth hormone, and leptin (O’Shea, and Gadina et al. 2002). Upon ligand binding, the receptor changes conformation, causing JAK2 to become activated through auto- and trans-phosphorylation at tyrosines 1007/1008 (Leonard and O’Shea, 1998; Yamaoka et al., 2004; Brown et al.,

2005). Once activated, JAK2 phosphorylates tyrosine residues on the

intracellular domain of the receptor creating docking sites for SH2 or phosphotyrosine- binding domains (Uhlik et al., 2005). Many proteins are involved in JAK2 mediated signaling cascades including signal transducers and activators of transcription (STATs), mitogen-activated protein kinases (MAPKs), protein kinase C, and phosphatidylinositol 3- kinase (PI3K) (Gouilleux et al., 1994; Wakao et al., 1994; DaSilva et al., 1996; Das et al.,

1996; Walters and Rillema, 1989; Berlanga et al., 1997).

STATs exist in an inactive state in the cytoplasm and are recruited to the receptor-

JAK2 complex through their SH2 domain. Once at the receptor, STATs become tyrosyl phosphorylated by JAK2 and form homo- or heterodimers (Darnell et al., 1994). These dimers then translocate to the nucleus where they interact with regulatory DNA elements and induce target gene transcription (Darnell et al., 1994). Different STATs are activated by different cytokines, for example prolactin activates STATs 1, 3, 5a and 5b while interferon-γ activates STAT1 and STAT5 (Dasilva et al., 1996; Gouilleux et al., 1994;

Sekimoto et al., 1997). These STATs form homo- or heterodimers and translocate to the nucleus and induce gene transcription (Ball et al., 1988). In addition to the STAT proteins, active JAK2 can also interact with phosphatases, adapter proteins Grb2, p85 phosphatidylinositol 3-kinase, SHC, and Cbl leading to activation of the phosphatidylinositol 3-kinase/Akt and Ras/Raf/ERK pathways (Ingley and Klinken,

2006;.Jatiani, Baker et al. 2010). Activation of STAT5 by prolactin-activated JAK2 plays a role in mammary turmorigenesis (Cotarla et al., 2004; Nevalainen et al., 2004; Sakamoto et al., 2010). JAK2 kinase activity is tightly regulated through auto-inhibition or by 3

suppressor of cytokine signaling (SOCS). SOCS proteins use various ways to inhibit JAK2 signaling, including competing for binding sites at the receptor and directly binding and inhibiting JAK2 activity (Argetsinger et al., 2004; Starr et al., 1997; Witthuhn et al., 1993).

JAK2 activity is also regulated by tyrosine phosphatases and the pseudokinase domain of

JAK2 (Shuai, 2003; Silvennoinen, 2002; Saharinen, 2000).

The JAK family of non-receptor protein tyrosine kinases mediates transduction of signals from cytokine receptors playing a key role in hematopoiesis. Mutations that alter the function of JAK kinases have been discovered in various malignancies of the hematopoietic system. Translocations including BCR-JAK2, TEL-JAK2 and PCM1-JAK2, can lead to constitutively active JAK2 in a variety of hematopoietic malignancies (Reiter et al. 2005; Bousquet et al. 2005; Murati et al. 2005). Gain- of-function mutations, including

V617F, JAK2 L611S, K539L mutant, T875N substitution in JAK2, and a ﬁve-amino-acid in-frame deletion (JAK2DeltaIREED) have been identiﬁed in myeloproliferative diseases

(James et al. 2005; Kralovics et al. 2005; Baxter et al. 2005; Levine et al. 2005; Mercher et al., 2006; Kratz et al., 2006; Malinge et al., 2007). Even though myeloproliferative disease pathogenesis has been extensively studied, it remains unclear how one point mutation leads to a variety of diseases.

JAK2 is expressed in most tissues and is thought to be located predominantly at the plasma membrane, where it associates with cytokine receptors (Behrmann et al., 2004).

However, JAK2 subcellular localization is still under debate. In addition to localizing at the plasma membrane, studies have identified JAK2 localizing to the cytoplasm, endoplasmic reticulum (ER) and nucleus (Ram and Waxmann, 1997). At the ER, JAK2 phosphorylates 4

ATPase p97, regulating assembly of the transitional ER (Lavoie et al., 2000). Nuclear localization of JAK2 has been seen in multiple cell types and causes phosphorylation of histone H3 leading to gene expression (Dawson et al., 2009). Additionally, JAK2 is associated with the transcription factor, NF1-C2, and prevent proteasomal degradation of

NF1-C2 (Nilsson et al., 2006). Regardless, nuclear localization of JAK2 is still under debate. JAK2 can also associate with microtubules and directly phosphorylate both α- and

β-tubulin, subunits of microtubules. In addition, tubulin can interact with STAT1, suggesting that tubulin is not only a substrate of JAK2 but mediates JAK2/STAT1 dependent signaling (Ma et al., 2007). Microtubules are highly dynamic polarized filaments composed of α-tubulin and β-tubulin dimers (Belmont et al, 1990; Verde et al,

1992). The plus end is more dynamic allowing for the addition of tubulin heterodimers while the minus end is less dynamic and is anchored and stabilized at microtubule organizing centers (Desai & Mitchison, 1997; Dammermann et al, 2003).

Centrosome

The centrosome is composed of a pair of centrioles surrounded by pericentriolar material (PCM, see Figure 2) (Paintrand et al., 1992; Bornens, 2002). The centrioles consist of nine microtubule triplets that form a symmetric cylinder. At the distal end of the centriole there is a transition zone where the microtubule triplets become doublets

(reviewed by Azimzadeh and Marshall, 2010). These microtubule doublets are linked together by an array of fine fibers (Paoletti et al., 1996; LeDizet et al., 1998; reviewed by

Salisbury, 2007). In mammalian cells, centrioles are 400nm in length and have a diameter of 200nm (Bornens, 2002; Azimzadeh and Bornens, 2007). The PCM is an electron dense

matrix that contains many proteins, including γ-tubulin ring complexes and ninein, which form a molecular scaffold to promote microtubule anchoring and nucleation (Choi et al,

2010; Dictenberg et al, 1998; Doxsey et al, 1994; Fong et al, 2008; Gomez-Ferreria et al,

2007; Zhu et al, 2008). At the onset of mitosis, the PCM dramatically

Figure 2. Centrosome Structure. The centrosome is a small organelle, usually 1-2µm in size, and is composed of a pair of centrioles made up of nine microtubule triplets. The more mature, mother, centriole contains a set of distal and subdistal appendages which are required for ciliogenesis and microtubule anchorage, respectively. Ninein mediates microtubule anchorage at the mother centriole. Both centrioles are connected by a fibrous linker containing rootletin which is anchored by C-Nap1 at the proximal end of each centriole. Pericentriolar material (PCM) surrounds the centriole pair, which contains γ- TURC complexes that nucleate microtubules (modified from Doxsey, 2001).

increases in size, allowing for an increase in microtubule nucleation at the centrosome

(Khodjakov & Rieder, 1999; Palazzo et al, 2000; Piehl et al, 2004).

The centriole pair contains a more mature mother centriole that contains two sets of appendages, distal and sub-distal, and the centriole which lacks these structures is the daughter centriole (see Figure 2). The distal appendages are required for ciliogenesis, and the subdistal appendages are required for microtubule anchorage at the centrosome

(Paintrand et al., 1992). Several proteins, including ninein, ε-tubulin, Cep164, and Cep170, have been identified as components of the subdistal appendages (Mogensen et al., 2000;

Chang et al., 2003; Guarguaglini et al., 2005; Ishikawa et al., 2005; Graser et al., 2007;

Soung et al., 2009). The centriole pair is tethered together at their proximal ends by a fibrous linker, keeping the duplicating centrioles in close proximity until the onset of mitosis. Dissociation of this linker is mediated by the activity of Nek2 kinase (reviewed by

Meraldi and Nigg, 2002). Nek2 is a centrosomal kinase that is regulated by the cell cycle.

At the end of G2 phase, PP1α, an inhibitor of Nek2, is inhibited by Cdk1, causing an increase in Nek2 kinase activity (Helps et al., 2000). This leads to phosphorylation of C-

Nap1 and Rootletin, components of the fibrous linker (Mayor et al., 2000; Bahe et al.,

2005). Once phosphorylated, C-Nap1 and rootletin are displaced from the centrosome, allowing the two centrosomes to separate (Fry et al., 1998; Bahe et al., 2005).

The centrosome is a non-membranous organelle found in close proximity to the nucleus and Golgi apparatus (reviewed by Doxsey, 2001). Traditionally, the centrosome is the microtubule organizing center of the cell (MTOC) (Robbins, 1968). In interphase cells, the centrosome recruits multiple proteins to the PCM, which allow for the concentrated microtubule nucleation and anchoring at the centrosome (Bobinnec et al., 1998;

Bettencourt-Dias and Glover, 2007; Nigg and Raff, 2009; Bornens, 2012). In addition to organizing interphase microtubules, centrosomes play a critical role in cell shape, polarity and motility. In mitotic cells, the centrosome contributes to spindle formation and plays a critical role in spindle positioning, which is critical for proper chromosome segregation

(Segal and Bloom, 2001; Doe and Bowerman, 2001; Schuyler and Pellman, 2001).

Centrosome duplication happens once per cell cycle, coordinating with cell-division

(see Figure 3). In new cells, there is a pair of centrioles tightly connected by a linker, referred to as S-M linker (Nigg and Sterns, 2011). This linker is formed during procentriole formation in S phase, allowing formation of one procentriole per mother and daughter centriole. Disengagement of this linker is critical for initiating centriole duplication (Tsou and Stearns, 2006). Upon centriole disengagement, the centriole pair is loosely linked together by a fibrous linker in early G1 phase (Fry et al., 1998; Mayor et al., 2000). At the onset of S phase, the procentriole forms perpendicularly at the proximal ends of the mother and daughter centriole. The procentrioles then elongates throughout S and G2 phase

(Vorobjev and Chentsov, 1982; Kuriyama and Borisy, 1981; Chretien et al., 1997). In G2 phase, centrosome maturation occurs, where there is an increase in PCM size and microtubule nucleation capacity in order to form two

Figure 3. Centrosome Duplication. Centrosome duplication is highly regulated, happening once per cell cycle. In early G1 the mother and daughter centriole undergo disengagement, and are loosely linked together at their proximal ends. At the onset of S phase, a procentriole forms at the proximal end of each centriole and elongates through G2. In G2 phase, centrosome maturation occurs, leading to an increase in PCM size and microtubule nucleation capacity. At the onset of mitosis, disassembly of the fibrous linker occurs, allowing the two centrosomes to separate and form the mitotic spindle (modified from Chen et al., 2003).

functional MTOCs (Palazzo et al, 2000). At the onset of mitosis, the fibrous linker disassembles allowing the centrosomes to separate and form the mitotic spindle (Fry et al.,

1998).

In addition to MTOC functions, further studies show that the centrosome functions as a cell cycle regulator involved in mitosis, anaphase onset, cytokinesis and G1 to S

transition (Sluder, 2005). Cdk1 is a mitotic kinase that is localized and activated at the centrosomes and promotes nuclear envelope breakdown, chromosome condensation and spindle formation (Jackman et al., 2003). Upstream regulators of Cdk1, Aurora-A and

Chk1, also associate with the centrosome (Jackman et al., 2003). Removal of centrosomes through micro-surgery resultes in cells that failed to complete cytokinesis, suggesting the centrosome plays a role in delivering regulatory components required for cell separation

(Piel et al., 2001). These data together propose an idea that the centrosome acts as a scaffold for cell cycle regulatory proteins, increasing the efficiency of cellular signaling pathways (Doxsey et al., 2005a; Doxsey et al., 2005b). Through the use of proteomic studies, hundreds of proteins have been identified to associate with the centrosome, including kinases, phosphatases, and cyclins supporting the role of centrosome as a scaffold for regulatory proteins (Anderson et al., 2003; Jakobsen et al., 2011). It is evident that centrosome function is more complex and extends far beyond its traditional role, which is made possible by the intricate structure and associated proteins of the centrosome.

Ninein

Ninein is a coiled-coil centrosomal protein first identified in mouse and later in humans. Human and murine ninein have highly conserved sequences with 70% identity and both contain a GTP-binding site, a large coil-coiled domain with four leucine zippers and an EF-hand-like domain (Figure 4) (Bouckson-Castaing et al. 1996). The coiled-coil

Figure 4. Ninein Structure. Ninein contains an EF-hand domain, GTP binding site, a large coiled-coil domain, and a GSK3β binding site. The three C-terminal leucine zippers are required for ninien localization to the centrosome. Ninein is able to bind to GSK3β through its C-terminal end. Even though exact sites of phosphorylation have not been

identified yet, ninein is phosphorylated by PKA, Aurora A and GSK3β at its C-terminal end (modified from Hong et al., 2000).

domain can form inter- and intra-molecular interactions (Chen et al., 2003). Ninein centrosomal localization requires the three leucine zippers located in the large coiled coil domain at the C-terminal end and recruits gamma tubulin through its N-terminal end

(Delgehyr et al., 2005; Lin et al., 2006). Four isoforms of human ninein (hNinein) and two isoforms of mouse ninein have been identified (Delgehyr et al., 2005). In hNinien, isoforms 1, 2 and 5 localize to the centrosome and can be phosphorylated by glycogen synthase kinase-3β (GSK3β), Aurora A and protein kinase A (PKA) (Howng et al., 2004;

Chen et al., 2003; Lin et al., 2006). However, isoform 6 is distributed around the perinuclear material and can be phosphorylated by PKA and Aurora A (Lin et al., 2006).

These data suggest that hNinein is a substrate to various kinases but the significance of phosphorylation by these kinases is still unknown. However, phosphorylation of ninein by

GSK-3β can be blocked by a novel ninein interaction protein, CCG1-99 (Howng et al.,

2004; Chen et al., 2003).

Through the use of immuno-electron microscopy it was revealed that ninein localizes to the subdistal appendages of the mother centriole and to the proximal end of both centrioles where it colocalizes with C-Nap1 (Mogensen et al., 2000; Piel et al., 2000;

Ou et al., 2002). Ninein localization to the centrosome is cell-cycle dependent (see Figure

5). Ninein localizes to the mother centriole in G1 and S phase; however, by G2 phase, ninein localizes to both mother and daughter centriole (Piel et al., 2000; Morgensen et al.,

2000; Ou et al., 2002). Centrosomal ninein is lost in mitotic cells, until late telophase, where ninein once again localizes to the mother centriole (Ou et al., 2002; Chen et al.,

2003).

Ninein plays a role in microtubule minus-end anchorage and serves as a docking site for the γ-tubulin ring complexes at the centrosome (Delgehyr et al., 2005; Stillwell et al.,

2004). Overexpressed ninein accumulates at the centrosome, decreasing microtubule release (Abal et al., 2002), and depletion of ninein causes a loss in microtubule anchorage at the centrosome (Abal et al., 2002; Dammermann and Merdes, 2002). It has also been reported that overexpressed ninein also causes Golgi fragmentation, a requirement for entry into mitosis, suggesting ninein may regulate cell progression into mitosis (Lin et al., 2006).

Increased cytoplasmic ninein is seen in polarized epithelial cells and neurons where the majority of microtubules are anchored at non-centrosomal apical sites (Mogensen et al.,

2000; Mogensen et al., 2002). Cytoplasmic distribution of ninein is dependent on microtubules and therefore, destabilization of microtubules causes a

Figure 5. Ninein localization during cell cycle. Ninein (red) localizes to the subdistal and proximal end of the mother centriole in G1 and S phase. By G2, ninein localizes to both the mother and daughter centriole. Ninein is not detectable during mitosis until, late telophase/early cytokinesis, where ninein localizes to the mother centrioles (modified from

Chen et al., 2003).

decrease in cytoplasmic ninein. Additionally, ninein is released from the centrosome in polarized epithelial cells, moves along the microtubules, and translocates to non- centrosomal apical sites where it colocalizes with β-catenin. However, it is unknown how centrosomal ninein is released (Moss et al., 2007).

Ninein is essential in early formation and patterning in the brain and knockdown of 13

ninein results in brain and skull deficiencies which are similar to phenotypes seen in

Microcephalic Primordial Dwarfism (Dauber et al., 2012). Additionally, knockdown of ninein in mouse models cause premature depletion of radial glia progenitors and increases in differentiating neurons. These data taken together implies ninein may play an essential role in neuron development (Dauber et al., 2012).

Chapter Two

Material and Methods

Plasmids - cDNAs encoding wild-type JAK2 (JAK2 WT) and constitutively active JAK2

V617F (both in pCDNA3 vector) were gifts of M. G. Myers (University of Michigan). The cDNA encoding GFP-JAK2 (in pEGFP-C1 vector) was a gift from L. J. Huang (University of Texas). The cDNA encoding Myc-tagged SH2B1β was a gift from C. Carter-Su

(University of Michigan). cDNAs encoding glutathione S-transferase (GST)-tagged truncated forms of mouse ninein (amino acids 1-246, 1-496, and 246 to 496)

(GenBank/EMBL/DDJB accession number AY515727) were gifts from M. Bornens

(Institute Curie, France). cDNAs encoding GFP-tagged WT and mutant ninein (amino acids 1179 to 1931) and His-tagged mutant ninein (amino acids 1617 to 2090) (human isoform 5; accession number AAG33512.2) were gifts from Y. R. Hong (Kaohsiung

Medical University, Taiwan).

Antibodies - Polyclonal anti-p1007/1008 Tyr-JAK2 (44426G) and monoclonal anti-JAK2

(Invitrogen), XP monoclonal rabbit anti-JAK2 (D2E12) (Cell Signaling), polyclonal anti-

JAK1 and anti-Tyk2 (Santa Cruz Biotechnology, Inc.), monoclonal anti-γ-tubulin (GTU-

88) (Sigma-Aldrich), polyclonal anti-γ-tubulin (Santa Cruz Biotechnology, Inc.), monoclonal anti-γ-tubulin (E7) (Developmental Studies Hybridoma Bank at the University of Iowa), polyclonal anti-centrin (H-40) (Santa Cruz Biotechnology, Inc.), polyclonal anti- phospho-histone H3 (Ser10) (Cell Signaling), monoclonal anti-phosphotyrosine (anti-PY)

(clone 4G10; EMD Millipore). Rabbit monoclonal anti-phospho-STAT1 (Tyr701) (D4A7) and anti-phospho-STAT1 (Tyr701) (58D6) were from Cell Signaling. Rabbit polyclonal 15

anti-STAT1 antibodies were from StressGen Biotechnology and both monoclonal rabbit anti-phospho-STAT5 (Tyr694) (C11C5) and polyclonal rabbit anti-STAT5 were from Cell

Signaling. Polyclonal anti-GST conjugated to horseradish peroxidase (HRP) and monoclonal anti-His were from Santa Cruz Biotechnology, Inc. Rabbit polyclonal anti- ninein (BioLegend) was used for immunoblotting and immunoprecipitation, and goat polyclonal anti-ninein (Santa Cruz Biotechnology, Inc.) was used for immunocytochemistry. Secondary goat anti-mouse IgG–Alexa Fluor 594, donkey anti- goat IgG–Alexa Fluor 647, and goat anti-mouse IgG–Alexa Fluor 488 antibodies were from Invitrogen.

Cells - 293T, T47D and COS-7 cells were purchased from American Type Culture

Collection (ATCC). 293T cells were grown in Dulbecco’s modification of Eagle’s

(DMEM) medium (Mediatech Cellgro) supplemented with 10 % calf serum, 1 mM L- glutamine, 100 units/mL of penicillin, 100 mg/mL of streptomycin. COS-7 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 1 mM L-glutamine,

100 units/mL of penicillin, 100 mg/mL of streptomycin. T47D cells were maintained in

RPMI (Mediatech) supplemented with 10% FBS, bovine insulin (0.2 units/ml; Sigma-

Aldrich), 50 units/ml penicillin, 50 μg/ml streptomycin and supplemented with 4 mM L- glutamine. γ2A cells (human fibrosarcoma-derived cells that lack JAK2 expression) and

2C4 cells (syngeneic parental cells that express wild-type JAK2) were a gift from G. R.

Stark (Lerner Research Institute, Cleveland Clinic Foundation, OH) and were previously described (Watling et al., 1993; Kohlhuber et al., 1997). Cells were maintained in DMEM supplemented with 10% FBS, 1 mM L-glutamine, 100 units of penicillin per ml, 100 mg

of streptomycin per ml, and 400µg/mL G418. HeLa cells stably expressing green fluorescent protein (GFP)-labeled centrin-1 were a gift from A. Khodjakov (Wadsworth

Center, Albany, NY) and were maintained in DMEM supplemented with 10% FBS, 1 mM

L-glutamine, 100 units of penicillin per ml, 100 mg of streptomycin per ml.

Synchronization - HeLa cells stably expressing GFP-centrin were plated in regular culture media a day prior to treatment. The next day, cells were treated with 2mM thymidine for 19 hours, and then released from the thymidine block by washing twice with phosphosaline buffer (PBS: 10 mM sodium phosphate, pH 7.4, 140 mM NaCl) before adding regular culture media. 10 hours later, the cells were treated with 2mM thymidine for 17 hours and then released by washing twice with PBS and adding regular culture media to the cells. Cells were then collected every two hours from 0 hours to 16 hours, fixed with ice-cold 70% ethanol, stained with 50mg/mL propidium iodide containing

1mg/mL RNase A for 30 minutes, and then analyzed by fluorescence-activated cell sorting

(FACS). The percentage of cells in different cell cycle phases were calculated by gating

G1, S, and G2/M cell populations in CellQuest. After release, the timepoints with the maximum percentage of cells for each phase was 0 hours for G1, 6 hours for S and 10 hours for G2/M.

Immunocytochemistry – T47D cells were plated on coverslips at a density of 100,000 cells per coverslip, and serum deprived for 72 hours. The cells were then treated with or without 200ng/mL of prolactin (A. F. Parlov, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD) for 15 or 45 minutes, permeabilized with microtubule stabilizing buffer with Triton X-100 (MTSB-T: 0.1M PIPES [pH 6.9], 1mM

EGTA, 4M glycerol, and 0.5% Triton X-100) for 1 minute, added to MTSB (without

Triton X-100) for 2 minutes and then fixed in ice cold methanol (-20˚C) for 7 minutes.

Cells were rehydrated in 1X KB (10mM Tris [pH 7.5], 150mM NaCl, .1% BSA, and

0.002% NaN3) for 10 minutes, blocked in 2% human serum and stained for anti-γ- tubulin(1:1000) and anti-JAK2 (1:200 dilution) or anti-pJAK2 (1:100 dilution) for 45 minutes. After washing for 30 minutes, cells were incubated in anti-mouse IgG–Alexa

Fluor 488 (1:200 dilution) with anti-rabbit IgG–Alexa Fluor 594 (1:200 dilution) for 45 minutes. For phosphotyrosine staining, the protocol above was repeated and cells were stained with anti-PY (1:200 dilution) and anti-γ-tubulin for 45 minutes. DNA was stained with 4’, 6’-diamindino-2phenylindole (DAPI, 1:3000 dilution, from Invitrogen). To confirm JAK2 centrosomal localization, T47D cells were plated on coverslips at a density of 100,000 cells per coverslip and transiently transfected with either 1µg GFP-JAK2 or

1µg GFP using Lipofectamine LTX, according to manufactures instructions, and 48 hours later cells were permeabilized and fixed as mentioned above. Cells were stained with anti-

γ-tubulin (1:1000) primary antibody for 45 minutes, anti-mouse IgG–Alexa Fluor 594

(1:200) secondary for 45 minutes and then DNA was stained with DAPI. Coverslips were mounted onto slides using ProLong© Gold (Invitrogen) antifade mountant and allowed to cure overnight. To assess centrosomal localization of either GFP WT ninein or GFP 1179-

1931 ninein (C-ninein) mutant, T47D cells were transiently transfected with 0.75µg C- ninein, 1µg GFP-WT ninein, or 1µg GFP with Lipofectamine© LTX. Then 48 hours after transfection, cells were permeabilized with MTSB-T, washed with MTSB and fixed with methanol, as mentioned above. Cells were stained with anti-γ-tubulin (1:1000) primary antibody and anti-mouse IgG–Alexa Fluor 594 (1:200) secondary. To assess whether 18

overexpression of WT or C-ninein alters the microtubule network, T47D cells were transfected with GFP WT ninein, C-ninein or GFP, permeabilized and fixed as mentioned above, and stained with anti-β-tubulin (1:500) primary and anti-mouse IgG–Alexa Fluor

594 secondary (1:400).

HeLa cells stably expressing GFP-centrin were plated at a density of 50,000 cells per coverslip. The next day, the cells were synchronized as mentioned above for G1, S and

G2/M phase. To obtain more mitotic cells, coverslips were fixed at 11.5 hour time point after release from the double thymidine block. At the proper time points, cells were permeabilized with 0.5% Triton X-100 in MTSB for 1 minute, added to MTSB for 2 minutes and then fixed in ice cold (-20˚C) for 7 minutes. Cells were then rehydrated in 1x

KB for 10min, incubated for 30 minutes in 2% human serum blocking buffer, followed by sequential staining beginning with anti-ninein (1:800 dilution) for 45 minutes. Coverslips were washed for 30 minutes followed by secondary anti-goat IgG–Alexa Fluor 647 (1:500 dilution) for 45 minutes. After washing for another 30 minutes, cells were incubated in anti-pJAK2 (1:100) for 45 minutes, washed for 30 minutes, and incubated in anti-rabbit

IgG–Alexa Fluor 594 (1:200 dilution) for 45 minutes, and then washed for 30 minutes.

DNA was stained with DAPI (from Invitrogen). Mitotic cells were categorized based on cell morphology, centrosomal separation, and DNA staining. Coverslips were mounted onto slides using ProLong© Gold antifade mountant and allowed to cure overnight.

Confocal imaging was performed with an inverted Leica TCS SP8 laser scanning confocal microscope using a 63x/1.4 numerical aperture (NA) objective lens. Sequential stacks with .1µm step interval were taken. All confocal images are maximal-intensity

projections.

Quantification of fluorescence intensity in digital images - Images of the centrosome were obtained with a 63x /1.4 NA objective lens. Fluorescence intensity at the centrosome was determined on digital images. Imaging conditions were identical for all cell cycle stages and images were acquired under conditions that ensured saturation was not reached.

Background was subtracted from the original images before quantification was performed in MetaMorph software. A region of interest (ROI) was drawn around the centriole

(centrin stain) and transferred to the corresponding pJAK2 and ninein z-stacks. The ROI was used to calculate average fluorescence intensity levels through the z-stack of pJAK2, ninein, and centrin stains for each stage of the cell cycle.

Microtubule Regrowth Assay – 48 hours after 15,000 γ2A and 20,000 2C4 cells were plated on coverslips, cells were treated with 5µM nocodazole and incubated at 4°C and

37°C for 30 minutes and 1 hour, respectively. Cells were then brought to room temperature and washed with warm Dulbecco’s Phosphate-Buffered Saline (DPBS).

Warm media was then added and microtubules were allowed to re-grow at 37°C. For the following times: 0, 1, 2.5, 5, 7.5, 10 minutes, cells were fixed with cold methanol at -20ºC for 10 minutes. Cells were blocked using 2% goat serum for 15 minutes and then stained with a 1:500 dilution of β-tubulin antibody (E7-Developmental Studies Hybridoma Bank) for 45 minutes. After washing cells for another 15 minutes, a 1:400 dilution of donkey anti-mouse IgG–Alexa Fluor 594 (invitrogen) secondary was used to stain cells for 45 minutes. Cells were then washed, mounted onto slides and visualized using a fluorescent microscope. For rescue experiments, γ2A (JAK2-null) cells were transfected with 3µg

JAK2 WT/pCDNA3 or JAK2 V617F/pCDNA3 using Lipofectamine LTX (Invitrogen) according to the manufacturer’s instructions. 48 hours after transfection the microtubule regrowth assay was performed as described above. Cells were stained with anti-β-tubulin and polyclonal JAK2 (1:400) for 45 minutes and then donkey anti-mouse IgG–Alexa Fluor

594 (1:400) and goat anti-rabbit IgG–Alexa Fluor 488 (1:200) secondary antibodies for 45 minutes. To assess microtubule nucleation, polymerization, and microtubule network formation, 100 cells were counted for each time point, only including JAK2 positive cells.

For JAK2 silencing, COS-7 cells were transfected with 200 nM JAK2 siRNA (Cell

Signaling) or 200 nM control siRNA-fluorescein isothiocyanate (FITC) (Cell Signaling) using the Lipofectamine RNAiMax reagent (Invitrogen) according to the manufacturer’s instructions and 48 hours later the regrowth assay was performed. JAK2 RNAi treated cells were stained with a 1:500 dilution of anti-β-tubulin antibody and polyclonal anti-

JAK2(1:100) for 45 minutes and then incubated with donkey anti-mouse IgG–Alexa Fluor

594 (1:400) and goat anti-rabbit IgG–Alexa Fluor 488 (1:200) for 45 minutes. For scramble-FITC RNAi treated cells, the secondary antibodies used were donkey anti-mouse

IgG–Alexa Fluor 594 (1:400) and goat anti-rabbit IgG–Alexa Fluor 647 (1:200). Cells were visualized using a fluorescent microscope. To assess microtubule nucleation, polymerization, and microtubule network formation, 100 cells were counted for each time point, only including JAK2 null cells.

To confirm that sites of microtubule nucleation occurred at the centrosome, the microtubule regrowth assay was performed with 2C4 and γ2A cells for the 1 and 2.5 minute timepoints. The cells were doubled stained with either monoclonal anti-β-tubulin

(1:500) and polyclonal anti-centrin (1:50) or anti-γ-tubulin (1:1000) and polyclonal centrin

(1:50). Donkey anti-mouse IgG–Alexa Fluor 594 and goat anti-rabbit IgG–Alexa Fluor

488 antibodies were used and the cells were visualized using a fluorescent microscope.

Assessment of the mitotic defects - 2C4 and γ2A cells were plated on poly-L-lysine- coated coverslips and synchronized by double thymidine blocking (see above). Cells were released for 10 hours and then were either fixed with cold methanol at 20°C for 10 min or treated with proteasome inhibitor MG132 (25 µM) (EMD) for an additional 1 h to arrest cells at metaphase and fixed. Fixed cells were stained with DAPI and anti-phospho-histone

H3 (only anti-phosphohistone H3-positive cells were scored) and scored for the appearance of mitotic errors. Abnormalities observed include misaligned metaphase chromosomes.

Purification of GST tagged construct

GST, GST-Ninein 1-496, GST-Ninein 1-246, GST-Ninein 246-496, and His-Ninein 1619-

2090, were transformed into BL21 codon plus bacteria. Cultures were grown to an OD600 of 0.5-0.6 and then a final concentration of .2mM IPTG was added and cultures grew either for 2 hours at 37˚C (GST and STAT5) or overnight at 18˚C (ninein constructs). The cultures were spun down at 4,000 RPM for 30 minutes at 4°C and the pellets were lysed in

15ml PBST(16mM Na2HPO4, 4mM NaH2PO4, 150mM NaCl, 1% Triton-X100, pH 7.3) plus 1xHaltTM protease inhibitor cocktail (Thermo Scientific) per 1L bacterial pellet. After the lysate was put through the French press, it was centrifuged at 10,500 RPM for 30 minutes twice. For GST-tagged proteins, 2ml glutathione agarose beads was added to the supernatant and rotated for 2 hour at 4°C. The beads were then washed three times with 22

TBST and once with 50mM Tris Base (pH=8.0). The beads were added to column and

0.5ml of glutathione elution buffer (50mM Tris-Base [pH 8.0] and 10mM reduced glutathione) was added to the beads and after 20 minutes, two 250μl fractions were collected. This was then repeated using 1ml of elution buffer and after 20 minutes, it was collected in (4) 250μl fractions. Then 1ml elution buffer was added to the beads and collected into 2 - 500μl fractions immediately. For His-ninein 1619-2090, 1mL nickelenitrilotriacetic acid (Ni-NTA)-agarose beads was added to the supernatant and rotated for 3 hours at 4˚C. The beads were washed with PBST, PBST containing 500mM

NaCl and 20mM imidazole, PBST containing 500mM NaCl and finally with PBST. 1mL elution buffer (200mM imidazole, 500mM NaCl, 1% triton X-100, pH 8.0) was added to the beads and rotated for 20 minutes at 4˚C. Bradford Assay was used to measure the amount of protein in each fraction and fractions with the highest protein concentrations were loaded on a gel, along with BSA standards.

In Vitro Kinase Assay - The kinase assay was performed as previously described (Rui et al., 2000). 100nM of JAK2 was incubated with either GST (0.5µg), 1-496 ninein (8µg), 1-

246 ninein (5µg), 246-496 ninein (5µg), 1619-2090 ninein (12µg), or STAT5 (3µg) in kinase buffer (50 mM HEPES [pH 7.6], 5 mM MgCl2, 0.5 mM dithiothreitol, 100 mM

NaCl, 1 mM Na3VO4) containing aprotinin (10μg/mL), leupeptin (10μg/mL), and 20μCi of

[γ-32P]ATP for 30 minutes at 30°C. The reaction was stopped with the addition of 100mM

EDTA, and proteins were separated by SDS-PAGE and visualized by autoradiography followed by immunoblotting with anti-JAK2, anti-GST-HRP, and anti-His.

Coimmunoprecipitation and Immunoblotting – For double transfection, T47D or 293T

cells were transfected with 3µg WT JAK2 and either 0.5µg GFP, 5µg C-ninein using PEI

(3µl PEI: 1µg DNA) or Calcium Phosphate method, respectively. The next day both

T47D and 293T cell lines were serum deprived for 24 hours and then treated with 200 ng/ml prolactin for 20 min or 1,000 U/ml human IFN-γ for 45 min (gift from D. W.

Leaman, University of Toledo, OH), respectively. Anti-JAK2 serum (1:100 dilution) was added to cell lysates and incubated on ice for 2 hours and rotated for 1 hour after the addition of protein A-agarose beads. For single transfection, T47D and 293T cell lines were transfected with 0.5µg GFP, 5µg C-ninein or 15µg WT ninein using PEI or Calcium

Phosphate method, respectively. Cells were serum deprived for 48 hours, treated and

JAK2 was immunoprecipitated as described above. Proteins were separated by SDS-

PAGE followed by immunoblotting.

For ninein, 10 plates of either T47D and 293T cell lines were combined. Anti-ninein

(1:100; Biolegend) was added to pre-cleared lysates and incubated for 6 hours at 4˚C.

Then, protein-A agarose beads were added and rotated for 2 hours at 4˚C. Proteins were separated by SDS-PAGE followed by immunoblotting.

For ninein silencing, T47D or 293T cell lines were transfected with 200 nM ninein siRNA

(Santa Cruz Biotechnology, Inc.) or 200 nM control siRNA (Cell Signaling) using the

Lipofectamine© RNAiMax reagent (Invitrogen). 48 hours later cells were transfected again with ether ninein siRNA or control siRNA.

Centrosome Purification – Centrosome isolation was based on a method previously described (Moudjou and Bornens, 1994). Briefly, exponentially growing GFP-centrin

HeLa cells were incubated with culture medium containing 1 μg/ml cytochalasin D and 0.2 24

μM nocodazole for 1 hour at 37°C. The cells were collected in TBS, centrifuged at 2,600 rpm at 4˚C, re-suspended in TBS, centrifuged, and then re-suspended in 2 mL of TBS containing 8% sucrose (wt/wt). An additional 8 mL of lysis buffer (1 mM Hepes [pH 7.2],

0.5% Nonidet P-40, 0.5 mM MgCl2, 0.1% 2-mercaptoethanol with proteinase and phosphatase inhibitors) was added and incubated for 10min on ice. Lysates were then spun at 4,600rpm for 10 min, and the supernatant was filtered through a 50μm nylon mesh. The final concentration of Hepes was adjusted to 10 mM and 2 units/mL of DNase I was added. The mixture was incubated for 30 min on ice and then the lysate was underlaid with

60% sucrose solution (10 mM Pipes [pH 7.2], 0.1% Triton X-100, 0.1% β- mercaptoethanol containing 60% sucrose (wt/wt)). The mixture was centrifuged for 30 min at 9,200 rpm in order to sediment the centrosomes into the sucrose cushion. The bottom

3ml containing the centrosomes were loaded onto a discontinuous sucrose gradient consisting of 1000μl of 70%, 500μl of 50% and 500μl of 40% (wt/wt) solutions and centrifuged for 1hr at 120,000 × g. Fractions were collected from the bottom and then diluted in 1ml of 10mM Pipes buffer (pH 7.2). Centrosomes were recovered by centrifugation at 13,000 rpm for 10 minutes and denatured in SDS sample buffer.

Chapter Three

Results

Active JAK2 localizes to the mother centriole with ninein.

To study the subcellular localization of endogenous JAK2 tyrosine kinase, T47D human breast cancer cells were serum-deprived for 72 hours, fixed and stained with anti-

JAK2 and a centrosome marker, anti-γ-tubulin. We have shown that JAK2 tyrosine kinase localized to one of two centrioles marked by γ-tubulin in an asynchronous cell population

(Figure 6A). In order to determine the subcellular localization of activated JAK2 tyrosine kinase, T47D cells were serum-deprived and then treated with the hormone prolactin

(PRL). PRL treatment led to JAK2 autophosphorylation and subsequent activation, which is recognized by the anti-p1007/1008 tyrosine-JAK2 antibody. Autophosphorylated JAK2

(pJAK2) localized to one of the two centrioles marked by γ-tubulin 15 minutes after PRL treatment and disappears from the centriole 45 minutes after PRL treatment (Figure 6B).

To confirm these results were not an artifact of the phospho-JAK2 antibody, the experiment was repeated using a general anti-phosphotyrosine antibody (PY). The PY antibody recognized one of two γ-tubulin marked centrioles only in serum deprived cells treated with

PRL for 15minutes (Figure 6C). Overexpression of GFP-tagged JAK2 WT construct in

T47D cells confirmed JAK2 localization to the centrosome (Figure 6D). Since proteins can associate with the centrosome during specific cell cycle phases, we decided to assess centrosomal localization of pJAK2 throughout the cell cycle by synchronizing HeLa cells stably expressing GFP-centrin, a centriole marker. HeLa cells were synchronized using a

Figure 6. Active JAK2 tyrosine kinase localizes to the centrosome.

(A). T47D cells were serum deprived, fixed and labeled for total JAK2 (red), γ-tubulin (green), and DAPI (blue). Inserts are of enlarged centrosomes. Yellow indicates

colocalization in merged images. JAK2 localized to one of two centrioles. (B) T47D cells were serum deprived and then treated with or without 200 ng/ml prolactin (PRL) for the indicated times. Cells were then fixed and labeled for activated JAK2

(p1007/1008 Tyr JAK2; red), and γ-tubulin (green). Inserts are of enlarged centrosomes. Yellow indicates colocalization in merged images. Activated JAK2 localizes to one of two centrioles. Magnification, 63x. Scale Bars, 10µm. Jay et al.,

Jay et al., Mol Cell Biol (2015) 35, 111-131.

Figure 6 (continued). Active JAK2 tyrosine kinase localizes to the centrosome. (C). T47D cells were serum deprived and then treated with or without 200ng/ml prolactin (PRL) for 15 minutes. Cells were fixed and labeled for general phosphor-tyrosyl staining using anti-phosphotyrosine antibody (PY; red), γ- tubulin (green) and DAPI (blue). Inserts are of enlarged centrosomes (asterisks). Anti-PY recognizes one of two γ-tubulin marked centrioles. (D). T47D cells overexpressing either GFP-tagged JAK2 WT (green) or GFP alone were fixed and labeled for γ-tubulin and DAPI. Overexpressed GFP-JAK2 WT localized to the centrosome. Inserts are of enlarged centrosomes (asterisks). Scale Bars, 10µm. Jay et al., Mol Cell Biol (2015) 35, 111-131.

double thymidine block, collected every two hours, fixed and stained in order to be analyzed by fluorescence-activated cell sorting (FACS). The highest amount of cells in G1,

S and G2 phase were at 0, 6, and 10 hour, respectively (Figure 7). Synchronized HeLa cells were stained with anti-ninein, a mother centriole marker, and DNA was stained with DAPI.

Typical ninein staining is a three-dot ring structure around the mother centriole, one dot represents staining at the proximal end and the other two dots stain the subdistal appendages of the mother centriole (Bouckson-Castaing et al., 1996a; Mogensen et al.,

2000). In G1 and S phase, pJAK2 colocalized with ninein (Figure 8A, G1 & S). By G2 phase, there was an increase in ninein and pJAK2 signaling which associated with both diplosomes (Figure 8A, G2). To further investigate the extent of pJAK2 colocalization with ninein, vertical cross sections were taken from reconstructed centrosomes using laser scanning confocal microscopy. Localization was scored either “exact colocalization” or

“partial colocalization”. In exact colocalization, pJAK2 colocalized with ninein at the distal end of the centriole; however, in “partial colocalization”, pJAK2 localized to the distal end of the centriole with ninein and along the length of the centriole. Vertical sectioning revealed that the majority (63.5%) of centrosomes have exact colocalization of pJAK2 with ninein in G1 phase; however, by S and G2 phase half of the centrosomes demonstrated exact colocalization while the other half demonstrated partial colocalization (Figure 8B).

By early mitosis, ninein signaling declined and did not reappear until telophase, as shown previously; whereas pJAK2 was present on both centrosomes throughout mitosis and cytokinesis (Figure 8C). The fluorescence intensities of pJAK2, ninein and GFP-centrin were measured at different cell cycle phases. pJAK2 levels at the centrosome increased

Figure 7. Double thymidine block for synchronizing HeLa cells.

HeLa cells stably expressing GFP-centrin were treated with 2mM thymidine for 19 hours, released from the thymidine block, and then 10 hours later cells were treated with 2mM thymidine. 17 hours later, cells were released and collected at different timepoints (0 hours – 16 hours). Cells were stained with propidium iodide and analyzed by FACS. After release, the timepoints with the maximal percentage of cells in each phase was 0 hours for G1, 6 hours for S and 10 hours for G2/M (graphs outlined in black).

Figure 8. Localization of pJAK2 during the cell cycle. (A). HeLa cells stably expressing GFP-centrin (green) were synchronized by a double thymidine block. Cells were fixed and labeled for ninein (blue) and active JAK2 (pJAK2; red). Scale bar, 1µm. JAK2 colocalizes with ninein around the mother centriole. (B). The x-z profile of the centrosome showed partial colocalization of pJAK2(red) and ninein (blue), with GFP-centrin in green. Scale bar 1µm. Quantification of centrioles with partial or exact colocalization between ninein and pJAK2 was carried out. n=63 centrioles for G1 cells, 54 for S cells, and 60 for G2 cells. Magnification, 63x. Z-step = .1µm. Jay et al., Mol Cell Biol (2015) 35, 111-131.

Figure 8 (continued). Localization of pJAK2 during the cell cycle. (C). HeLa cells stably expressing GFP-centrin (green) were synchronized by a double thymidine block. Cells were fixed and labeled for ninein (blue) and active JAK2 (pJAK2; red). Ninein disappears from the centrosome during mitosis and reappears in telophase. pJAK2 was present at the mother centrioles during mitosis and colocalizes with ninein on the mother centriole during cytokinesis. Scale bar, 8µm. Magnification, 63x. Z-step = .1µm. Jay et al., Mol Cell Biol (2015) 35, 111-131.

through the cell cycle, peaked at S/G2, decreased in prophase, and peaked again in prometaphase; whereas, ninein levels decreased beginning in prophase and returned to staring (G1) levels during cytokinesis. GFP-centrin stayed constant throughout the cell cycle phases (Figure 8D). These data taken together demonstrated that pJAK2 is centrosomal component that colocalizes with ninein at the mother centriole.

JAK2 plays a role in microtubule regrowth at the centrosome.

Since pJAK2 localized with ninein, a microtubule anchoring protein, at the centrosome, we decided to test whether JAK2 plays a role in centrosomal microtubule dynamics. Even though microtubule nucleation can occur at non-centrosomal sites, the centrosome is the major site of microtubule nucleation in mammalian cells. In interphase mammalian cells, cytoplasmic MT may be organized in two different ways: (i) in radial MT arrays anchored at the centrosomes (MT are released, recaptured, and anchored at the centrosome) or (ii) in the noncentrosomal nonradial network (MT are released from centrosome into the cytoplasm) (Bartolini and Gundersen, 2006; Keating et al., 1997).

Multiple processes play an important role in microtubule organization in interphase cells, including nucleation, release, and anchoring. A microtubule regrowth assay was utilized to assess whether JAK2 plays a role in microtubule nucleation, release, or anchorage at the centrosome. COS-7 cells, which contain radial MT arrays anchored at the centrosomes, were depleted of JAK2 using siRNA, treated with nocodazole for 1.5 hours and then the nocodazole was washed out, to allow microtubule regrowth. The cells were fixed at different timepoints and then stained for JAK2 and microtubules. Cells were scored for the beginning of MT nucleation (bright anti-β-tubulin positive spots in 33

Figure 8 (continued). Localization of pJAK2 during the cell cycle. (D). Fluorescence intensities of GFP-centrin, ninein and pJAK2 at centrosomes during different cell cycle stages was quantified using MetaMorph. Bars Barsrepresent repre means ±SE. *, signiﬁcant at a P value of 0.024 for cells in S compared with cells in G1,a P value of 0.006 for cells in G2 compared with cells in G1, a P value of 0.000018 for cells in prophase compared with cells in G2, and a P value of 0.039 for cells in prometaphase compared with cells in prophase. Each experiment was repeated three times and more than 30 centrioles per cell cycle phase were analyzed. Graph represents average of all experiments. Jay et al., Mol Cell Biol (2015) 35, 111-131.

the cytoplasm) (Figure 9A, second cell image from the top), MT aster formation (Figure

9A, third cell image from the top), and the presence of a cytoplasmic MT network (Figure

9A, bottom). Control and JAK2 depleted cells initiated microtubule nucleation at the same rate (Figure 9A, top graph); however, by 2.5 minutes, microtubule organization in JAK2 depleted cells was already altered, where only 68% of the cells, contained radial microtubule network as compared to 90% in control cells (Figure 9A, middle graph).

JAK2-depleted cells contained a significantly higher amount of cells with randomly distributed cytoplasmic microtubules compared to control cells (Figure 9A, bottom).

Together these data suggest that microtubule anchoring but not nucleation was affected in

JAK2-depleted cells. We next tested whether JAK2 plays a similar role in cells in which microtubules are randomly distributed in the cytoplasm. We utilized two human HT1080 fibrosarcoma derived cell lines, 2C4 cells, syngeneic parental cells that express JAK2, and

γ2A cells that lack JAK2 expression (Kohlhuber et al., 1997; Watling et al., 1993). Both cell lines initiated microtubule nucleation at the same rate (Figure 9B, second image from top and top graph). The majority of 2C4 cells contained large microtubule asters by 2.5 minutes after washout; whereas, in γ2A cells, aster formation was delayed, peaking at 5 minutes (Figure 9B, third image and middle graph). In addition, microtubule release was delayed in γ2A cells compared to that in 2C4 cells by 5 minutes (Figure 9B, bottom image and bottom graph). Since microtubule nucleation can occur at non-centrosomal sites, we repeated the microtubule regrowth assay for the 1 and 2.5 minute timepoints. There were one or two bright β-tubulin positive spots in the cytoplasm of both cell lines, which were positive for anti-centrin and anti-γ-tubulin, confirming nucleation occurred at the centrosome (Figure 9C). These data taken together reveal that aster formation and 35

Figure 9. JAK2 regulates aster formation and microtubule release. (A). COS-7 cells were transiently transfected with either ninein siRNA or control siRNA and treated with 5µM nocodazole to depolymerize microtubules (top, 0 minutes). After nocodazole was washed out to allow microtubule regrowth, cells were fixed at 1, 2.5, 5, 7.5 and 10 minutes. Microtubules were labelled with anti-β-tubulin and anti-JAK2 and cells were scored for initiation of microtubule nucleation (top graph), aster formation (middle graph), and microtubule release (bottom graph). Only JAK2 negative cells were scored for JAK2 depleted cells. The solid line represents control COS-7 cells, and the dotted line represents JAK2-depleted cells. n = 3; 100 transfected cells were scored in each experimental condition. The bars represent means ±SE. Scale bars, 10 µm. Jay et al., Mol Cell Biol (2015) 35, 111-131.

Figure 9 (continued). JAK2 regulates aster formation and microtubule release. (B). 2C4 (control) and γ2A cells were treated with 5µM nocodazole to depolymerize microtubules (top, 0 minutes). After nocodazole was washed out to allow microtubule regrowth, cells were fixed at 1, 2.5, 5, 7.5 and 10 minutes. Microtubules were labelled with anti-β-tubulin and cells were scored for initiation of microtubule nucleation (top graph), aster formation (middle graph), and microtubule release (bottom graph). Cells were stained for anti-JAK2 and only JAK2 negative cells were scored for JAK2 depleted cells. The solid line represents 2C4 cells, and the dotted line represents γ2A cells. n = 6; 100 transfected cells were scored in each experimental condition. The bars represent means ±SE. Scale bars, 10 µm. Jay et al., Mol Cell Biol (2015) 35, 111-131.

Figure 9 (continued). JAK2 regulates aster formation and microtubule release. (C). 1 and 2.5 minutes after nocodazole washout γ2A cells were fixed and stained with anti-centrin (green) and either anti-β-tubulin (red) or anti-γ-tubulin (red). Microtubule nucleation starts at the centrosome in γ2A. Arrows indicate centrosomes which are enlarged in right merged images. Scale bar, 10µm. Jay et al., Mol Cell Biol (2015) 35, 111-131.

microtubule release were delayed in JAK2 null cells even though the absence of JAK2 did not completely disrupt microtubule nucleation, anchoring, and release. To confirm the results observed in γ2A cells were caused from the absence of JAK2, JAK2 WT and constitutively active JAK2 V617F were re-expressed in γ2A cells. Re-expression of either

JAK2 WT or V617F partially rescued aster formation and microtubule release, suggesting an involvement of JAK2 in controlling centrosomal microtubule anchorage (Figure 9D).

JAK2 tyrosyl phosphorylates N-terminal ninein (amino acids 1-246), while JAK2 kinase activity is inhibited by C-terminal ninein (1617-2090).

Ninein is protein involved in microtubule anchorage at the centrosome (Bouckson-

Castaing et al., 1996; Delgehyr et al., 2005; Mogensen et al., 2000). Since our data showed pJAK2 localizing with ninein at the centrosome and the fact that JAK2 regulates microtubule anchorage, we investigated whether ninein was a substrate of JAK2. In 293T and T47D cell lines, endogenous ninein co-immunoprecipitated with endogenous JAK2, along with the positive control, SH2B1β, a known JAK2 target (Figure 10A).

Subsequently, JAK2 co-immunoprecipitated with endogenous ninein, but not JAK1 or

Tyk2, demonstrating that the JAK2/ninein interaction is specific (Figure 10B). In addition, immunoprecipitated endogenous ninein was recognized by a general anti-phosphotyrosine antibody (Figure 10C). Furthermore, GFP, GFP-tagged WT ninein or GFP- tagged C- terminal ninein was overexpressed in 293T cells. Overexpressed WT and C-terminal ninein but not GFP alone coimmunoprecipitated with endogenous JAK2 (Figure 10D). In addition, centrosomes were isolated by a discontinuous sucrose-density gradient in HeLa cells stably overexpressing GFP-centrin. 39

Figure 9 (continued). JAK2 regulates aster formation and microtubule release. (D). For recovery experiments, JAK2 WT (top row) or JAK2 V617F (bottom row) was over expressed in γ2A cells and cells were treated with 5µM nocodazole to depolymerize microtubules (top, 0 minutes). After nocodazole was washed to allow microtubule regrowth, cells were fixed at 1, 2.5, 5, 7.5 and 10 minutes. Microtubules were labelled with anti-β-tubulin and anti-JAK2 and only JAK2 positive cells were scored for initiation of microtubule nucleation (left graph), aster formation (middle graph), and microtubule release (right graph). The solid line represents 2C4 cells, the dashed line represents γ2A cells, and the dotted line represents γ2A cells overexpressing either JAK2 WT (top row) or JAK2 V617F (bottom row). n=3 for JAK2 WT and n=7 for JAK2 V617F experiments. One hundred transfected cells were scored in each experiment for each condition. Jay et al., Mol Cell Biol (2015) 35, 111-131.

Figure 10. Endogenous JAK2 associates with endogenous ninein and WT and C-ninein. (A). T47D and 293T whole cell lysates were immunoprecipitated with anti-JAK2 or control IgG. The nitrocellulose was immunoblotted with the indicated antibodies. Endogenous SH2B1β was an internal control. (B). T47D and 293T whole cell lysates were immunoprecipitated with anti-ninein or control IgG. The nitrocellulose was immunoblotted with the indicated antibodies. γ-tubulin was an internal control. Endogenous JAK2, along with γ-tubulin, but not JAK1 or Tyk2 coimmunoprecipitated with ninein. Blots for immunoprecipitates and whole cell lysates (WCL) were from the same nitrocellulose. Experiments were repeated three times. T47D experiments performed by Alan Hammer. Jay et al., Mol Cell Biol (2015) 35, 111-131.

Figure 10 (continued). Endogenous JAK2 associates with endogenous ninein and WT and C-ninein. (C). Endogenous ninein was immunoprecipitated from 293T whole cell lysates. The nitrocellulose was immunoblotted with the indicated antibodies. A general phosphotyrosine antibody (anti-PY; clone 4G10) recognized endogenous ninein. (D). GFP-tagged WT ninein, GFP-tagged C-terminal ninein (amino acids 1179-1931), or

GFP alone were overexpressed in 293Tcells. Endogenous JAK2 was immunoprecipitated with anti-JAK2 or control IgG and the nitrocellulose was immunoblotted with indicated antibodies. All blots of the immunoprecipitates and whole cell lysates (WCL) came from the same nitrocellulose. Each experiment was repeated three times. Jay et al., Mol Cell Biol (2015) 35, 111-131.

JAK2 and ninein copurified in the same centrosomal fractions (Figure 11). These data demonstrate that JAK2 interacts with ninein. To further determine whether JAK2 directly phosphorylates ninein, different ninein fragments were utilized in an in vitro kinase assay.

Recombinant active JAK2 (amino acids 808 to 1132 of the WT protein) phosphorylated ninein fragments consisting of amino acids 1 to 496 (Fig. 12A, lane 2) and 1 to 246 (Fig.

12A, lane 3) but not ninein fragments consisting of amino acids 246 to 496 (lane 4) and

1617 to 2090 (lane 5) or GST alone (lane 1). These data demonstrate that the N-terminus of ninein (amino acids 1 to 246) is directly phosphorylated by JAK2. Therefore, we investigated the efficiency of JAK2 to phosphorylate ninein compared to STAT5, a known

JAK2 substrate, in an in vitro kinase assay. His- tagged recombinant STAT5 protein was highly phosphorylated by JAK2 compared to N-terminal ninein (Figure 12B).

Interestingly, even though JAK2 autophosphorylation levels were constant when incubated with N-terminal fragments of ninein (Figure 12A, lanes 2 to 4 in the upper image) or GST

(lane 1), incubation of JAK2 with C-terminal ninein completely inhibited autophosphorylation of JAK2 (lane 5). Therefore, we investigated whether JAK2 activity is inhibited by C-terminal ninein by incubating increasing amounts of His-tagged C-terminal ninein (amino acids 1617-2090) with equal amounts of recombinant JAK2 in an in vitro kinase assay (Figure 12C). These data demonstrate that His-ninein (amino acids 1617-

2090) inhibits JAK2 activity in a dose dependent manner. To examine the subcellular localization of GFP- tagged WT and C-terminal ninein, T47D cells were transfected with

GFP WT ninein, GFP C-terminal ninein, or GFP alone. Both WT and C-terminal ninein colocalized with γ-tubulin at the centrosome while GFP was diffused

Figure 11. Endogenous JAK2 copurified with endogenous ninein in the same centrosomal fraction. (A). Whole cell lysates from exponentially growing HeLa cells stably expressing GFP- centrin, were subjected to sucrose gradient fractionation. Fractions were collected from the bottom. The nitrocellulose was immunoblotted with the indicated antibodies. JAK2 and ninein copurified to the same fractions and both ninein and JAK2 were recognized by anti-phosphotyrosine antibody. The experiment was repeated three times.

Figure 12. Recombinant JAK2 tyrosyl phosphorylates the N-terminus of ninein while the C-terminus of ninein inactivates JAK2 activity in vitro. (A). Different ninein mutants were subjected to an in vitro kinase in the presence of recombinant active JAK2 fragment (amino acids 808-1132). The nitrocellulose was immunoblotted with the indicated antibodies. JAK2 phosphorylated ninein mutants containing amino acids 1-496 (lane 2) and amino acids 1-246 (lane 3) but not the mutant containing amino acids 246-496(lane 4) or amino acids 1617-2090 (lane 5) or GST alone (lane 1). JAK2 was autophosphorylated in each lane except lane 5 which was incubated with ninein amino acids 1617 to 2090. The experiment was repeated three times. Jay et al., Mol Cell Biol (2015) 35, 111-131.

Figure 12 (continued). Recombinant JAK2 tyrosyl phosphorylates the N- terminus of ninein while the C-terminus of ninein inactivates JAK2 activity in vitro. (B). GST-tagged ninein (amino acids 1-496), recombinant His-tagged STAT5, or GST alone were subjected to an in vitro kinase in the presence of recombinant active JAK2 fragment (amino acids 808-1132). The nitrocellulose was immunoblotted with the indicated antibodies. JAK2 phosphorylated STAT5 more efficiently than ninein (1-496). The experiment was repeated three times. Jay et al., Mol Cell Biol (2015) 35, 111-131.

Figure 12 (continued). Recombinant JAK2 tyrosyl phosphorylates the N- terminus of ninein while the C-terminus of ninein inactivates JAK2 activity in vitro. (C). Increasing amounts of His-tagged ninein (amino acids 1617-2090) or GST alone were subjected to an in vitro kinase in the presence of recombinant active JAK2 fragment (amino acids 808-1132). The nitrocellulose was immunoblotted with the indicated antibodies. JAK2 autophosphorylation decreased with increasing amounts of His-tagged ninein (1617-2090). ns, non-specific bands. The experiment was repeated three times. Jay et al., Mol Cell Biol (2015) 35, 111-131.

Figure 12 (continued). Recombinant JAK2 tyrosyl phosphorylates the N- terminus of ninein while the C-terminus of ninein inactivates JAK2 activity in vitro. (D). T47D cells were transfected with either GFP-tagged WT ninein, GFP-tagged C- terminal ninein (amino acids 1179-1931) or GFP alone, fixed and labelled for γ- tubulin (red). Both WT and C-ninein colocalized with γ-tubulin. Insets show enlarged centrosomes (marked by asterisks). DAPI stained the cell nucleus (blue). Scalebar,10µm. Jay et al., Mol Cell Biol (2015) 35, 111-131.

throughout the cytoplasm (Figure 12D). Depending on the level of GFP C-terminal ninein expression, there was formation of either small dots or large aggregates in the cytoplasm, previously described (Lin et al., 2006). Cells overexpressing GFP WT or C-terminal ninein were immunolabeled with β-tubulin to check for alterations in the microtubule cytoskeleton and neither WT nor C-terminal ninein altered the microtubule cytoskeleton. We next investigated whether C-ninein inhibited JAK2 activity in vivo. T47D cells were transfected with JAK2 and either GFP-tagged C-terminal ninein (amino acids 1179-1931), SH2B1β (a known JAK2 activator and positive control), or GFP vector alone and then treated with or without prolactin. JAK2 was immunoprecipitated and autophosphorylation of JAK2 was assessed by immunoblotting with anti-phosphotyrosine antibody. Upon prolactin treatment,

JAK2 became activated and overexpression of SH2B1β led to increased JAK2 tyrosyl kinase activity. However, overexpression of C-terminal ninein led to decreased JAK2 tyrosyl kinase activity (Figure 13A). This experiment was repeated in 293T cells treated with γ-interferon (IFNγ). Even though overexpressed JAK2 is constitutively active in 293T cells, treatment with IFNγ increased JAK2 phosphorylation (Jiao et al., 1996; Rui et al.,

1997). Overexpressed SH2B1β increased JAK2 tyrosyl kinase activity, while C-terminal ninein decreased JAK2 tyrosyl kinase activity. These results demonstrated that C-terminal ninein inhibits JAK2 tyrosyl kinase activity.

WT and C-terminal ninein reduce STAT1 and STAT5 tyrosyl phosphorylation.

Next we decided to investigate whether C-terminal ninein could inhibit signaling events downstream of JAK2 by studying STAT1 and STAT5 tyrosyl phosphorylation.

Figure 13. C-terminal ninein decreases JAK2 tyrosyl kinase activity in vivo. (A). T47D and 293T cells overexpressing JAK2 and either GFP-tagged C-terminal ninein (amino acids 1179-1931; lanes 2 and 5), adapter protein SH2B1β (JAK2 kinase activity enhancer, positive control, lanes 3 and 6) or vector (lanes 1 and 4) were serum deprived and treated without (lanes 1-3) or with (lanes 4-6) prolactin or γ-interferon, respectively. JAK2 was immunoprecipitated with anti-JAK2 and immunoblotted with the indicated antibodies. The graphs represent densitometric analysis of the bands obtained from phosphorylated proteins normalized with total proteins. Bars represent means ± S.E. *, signiﬁcant; P values are indicated for comparisons with cells expressing vector and with the same treatment. n=4. T47D work performed by Alan Hammer. Jay et al., Mol Cell Biol (2015) 35, 111-131.

T47D cells were transfected with JAK2 and either C-terminal ninein, SH2B1β or GFP alone and then treated with prolactin. Cell lysates were immunoblotted for phospho-

STAT5. Prolactin treatment led to STAT5 phosphorylation and overexpression of SH2B1β increased STAT5 tyrosyl phosphorylation, as expected (Figure 13B), while overexpressed

C-terminal ninein significantly reduced tyrosyl phosphorylation of STAT5. These results were repeated in 293T cells co-transfected with JAK2 and either C-terminal ninein,

SH2B1β or GFP alone. INFγ treatment led to STAT1 phosphorylation, and SH2B1β increased STAT1 phosphorylation further (Figure 13B). However, C-terminal ninein decreased INFγ stimulated STAT1 activation. STAT5 activation by IFNy is cell type specific (Burdeinick-Kerr et al., 2009; Meinke et al., 1996; Woldman et al., 2001), and,

INFγ induced STAT5 phosphorylation in 293T cells. Overexpressed SH2B1β increased

STAT5 phosphorylation while C-terminal ninein decreased STAT5 activation.

We next decided to investigate how WT and C-terminal ninein affected activities of endogenous JAK2 and STAT. T47D cells were transfected with either WT or C-terminal ninein and treated with prolactin. Prolactin treatment led to both JAK2 and STAT5 phosphorylation in control cells (Figure 14A). Expression of either WT or C-terminal ninein reduced endogenous JAK2 and STAT5 phosphorylation. Next, we overexpressed either WT or C-terminal ninein in 293T cells. IFNγ treatment promoted phosphorylation of endogenous JAK2 and subsequently endogenous STAT1 and STAT5 (Figure 14B).

However, expression of WT and C-terminal ninein reduced IFNγ induced phosphorylation of endogenous JAK2, STAT1 and STAT5. To confirm that ninein

Figure 13 (continued). C-terminal ninein decreases JAK2 tyrosyl kinase activity in vivo. (B). T47D and 293T cells overexpressing JAK2 and either GFP-tagged C-terminal ninein (amino acids 1179-1931; lanes 2 and 5), adapter protein SH2B1β (JAK2 kinase activity enhancer, positive control, lanes 3 and 6) or vector (lanes 1 and 4) were serum deprived and treated without (lanes 1-3) or with (lanes 4-6) prolactin or interferon-γ, respectively. Whole cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. The graphs represent densitometric analysis of the bands obtained from phosphorylated proteins normalized with total proteins. Bars represent means ± S.E. *, signiﬁcant; P values are indicated for comparisons with cells expressing vector and with the same treatment. n=3. T47D experiments performed by Alan Hammer. Jay et al., Mol Cell Biol (2015) 35, 111- 131. 52

Figure 14. WT and C-terminal ninein decrease endogenous JAK2 autophosphorylation and subsequently STAT1 and STAT5 tyrosyl phosphorylation. (A). T47D cells overexpressing WT ninein, C-terminal ninein, or vector were serum deprived and treated with or without 200ng/ml prolactin for 15 minutes. JAK2 was immunoprecipitated with anti-JAK2. The immunoprecipitates (top) and whole cell lysates (bottom) were resolved with SDS-PAGE and immunoblotted with the indicated antibodies. The graphs represent densitometric analysis of the bands obtained from phosphorylated proteins normalized with total proteins. Bars represent means ± S.E. *, signiﬁcant; P values are indicated for comparisons with cells expressing vector and with the same treatment. n=7. Experiments performed by Alan Hammer. Jay et al., Mol Cell Biol (2015) 35, 111-131.

Figure 14 (continued). WT and C-terminal ninein decrease endogenous JAK2 autophosphorylation and subsequently STAT1 and STAT5 tyrosyl phosphorylation. (B). 293T cells overexpressing WT ninein, C-terminal ninein, or vector were serum deprived and treated with or without 1000U/ml interferon-γ for 45 minutes. JAK2 was immunoprecipitated with anti-JAK2. The immunoprecipitates (top) and whole cell lysates (middle and bottom) were resolved with SDS-PAGE and immunoblotted with the indicated antibodies. The graphs represent densitometric analysis of the bands obtained from phosphorylated proteins normalized with total proteins. Bars represent means ± S.E. *, signiﬁcant; P values are indicated for comparisons with cells expressing vector and with the same treatment. n=4. Jay et al., Mol Cell Biol (2015) 35, 111-131.

decreases JAK2 activation, we used a ninein specific siRNA to knockdown endogenous ninein in T47D and 293T cells. Knockdown of ninein led to increased JAK2 and STAT5 tyrosyl phosphorylation as compared to control cells (Figure 14C). Taken together these data demonstrate that overexpression of either WT or C-terminal ninein inhibits JAK2,

STAT1 and STAT5 tyrosyl phosphorylation in response to different ligands.

JAK2 depletion causes mitotic defects.

Proper chromosome segregation is dependent on properly anchored microtubules and since our data demonstrated that JAK2 regulates microtubule anchorage, we decided to investigate whether JAK2 null cells contain mitotic defects. Both 2C4 and γ2A cells contained mitotic defects, however, γ2A contained significantly more defects than 2C4 cells (Figure 15). Specifically, around 21% of the metaphase 2C4 cells had defects in chromosome alignment (i.e., chromosome misaggregation), compared to 37% of the metaphase γ2A cells, suggesting that γ2A cells have defects in attachment of the chromosomes to the spindle microtubules. The most frequent mitotic defect was lagging chromosomes and anaphase bridges γ2A cells. Lagging chromosomes are a result of both spindle poles attaching to one kinetochore, caused by centrosome amplification (Ganem et al., 2009; Silkworth et al., 2009). However, when γ2A cells were immunostained for centrin or γ-tubulin (Figure 9C) there was no evidence of centrosome amplification and therefore chromosome lagging was not a result from centrosome amplification in these cells. Anaphase bridges are a hallmark of genomic instability in malignant cells and upon breakage, cause chromosomal rearrangements including deletions, translocations and deletions (Gisselsson et al., 2001; Hoffelder et al., 2004). 55

Figure 14 (continued). WT and C-terminal ninein decrease endogenous JAK2 autophosphorylation and subsequently STAT1 and STAT5 tyrosyl phosphorylation. (C). T47D and 293T cells were transfected with either control or ninein siRNA, serum deprived and then treated with prolactin or interferon-γ, respectively. Whole cell lysates were resolved with SDS-PAGE and immunoblotted with the indicated antibodies. Knockdown efficiency was judged by immunoblotting with anti-ninein. T47D experiments performed by Alan Hammer. Jay et al., Mol Cell Biol (2015) 35, 111-131.

Figure 15. Loss of JAK2 leads to mitotic errors. 2C4 and γ2A cells were synchronized by double thymidine block and then treat with or without MG132 (for assessment of metaphase cells only). Cells were stained with DAPI and and scored for mitotic errors. Scale bar 10µm. The graph depicts the percentage of cells containing chromosome misalignment (700 metaphase cells from 9 independent experiments), lagging chromosomes and anaphase bridges (300 anaphase cells from 6 independent experiments) and micronuclei (1,400 interphase cells from 4 independent experiments). Bars represent means ± S.E. *, signiﬁcant; P values are those for comparisons with 2C4 cells. Experiments performed by Dr. Maria Diakonova. Jay et al., Mol Cell Biol (2015) 35, 111- 131.

γ2A cells also contained a higher percentage of micronuclei around the interphase nucleus than 2C4 (3% in 2C4 and 8% in γ2A) and micronuclei are also suggestive of the generation of aneuploid cells (Rajagopalan and Lengauer, 2004). These data demonstrate that the absence of JAK2 results in mitotic defects which lead to aneuploidy and genomic instability. Therefore, it is critical to maintain normal levels of JAK2 in order to preserve the genome.

Loss of JAK2 affects centrosome cohesion.

Since pJAK2 localized to the centrosome independently of ninein during mitosis, we hypothesized that pJAK2 may participate in ninein independent functions, such as centrosome cohesion. Centrosomes stay in close proximity until late G2 phase, when certain centrosome proteins are phosphorylated to allow the centrosomes to separate

(Bornens, 2002; Meraldi and Nigg, 2002; Meraldi and Nigg, 2001). To investigate whether

JAK2 plays role in centrosome cohesion, 2C4 and γ2A cells were fixed and stained with anti-γ-tubulin and DAPI. 2C4 interphase cells contained centrosomes separated by 1-2µm in distance, whereas γ2A cells contained centrosomes separated by more than 2µm in distance (Figure 16A). Two widely separated centrosomes were observed in 34% of γ2A cells compared to only 12% in 2C4 cells (Figure 16B). To confirm that premature centrosome separation seen in γ2A cells was due to JAK2 deficiency, JAK2 WT or JAK2

V617F was re-expressed in γ2A cells. γ2A cells overexpressing JAK2 WT decreased the percentage of cells containing split centrosomes (14%) but there was no significant reduction in cells overexpressing JAK2 V617F (24%). Further evidence that JAK2 is important of centrosome cohesion was obtained in COS-7 cells depleted of JAK2. 58

Depletion of JAK2 in COS-7 cells resulted in a 2.7 fold increase in separated centrosomes when compared to control siRNA transfected cells (Figure 16C). Taken together, these data suggest that both loss and overactivation of JAK2 results in centrosome cohesion defects.

Figure 16. Loss of JAK2 causes centrosome splitting. (A) 2C4, γ2A, and γ2A cells overexpressing either JAK2 WT or constitutively active JAK2 V617F were labeled with anti-γ-tubulin and DAPI (2C4 and γ2A cells) or anti-γ-tubulin and anti-JAK2 (γ2A+JAK2 WT and γ2A+JAK2 V617F cells; only anti-JAK2-positive cells were scored). Interphase cells were scored for the appearance of split centrosomes. Arrows and arrowheads mark paired centrioles (1-2 µm apart) and split centrosomes (>2 µm apart), respectively.

Asters indicate transfected cells. (B) Centrosomes were counted as split when the distance between centrioles was > 2μm. (C) Quantification of centrosome splitting in COS-7 cells overexpressing either control siRNA-FITC (only FITC- positive cells were scored) or JAK2 siRNA (only anti-JAK2-negative cells were scored). Bars represent means ± S.E. *, signiﬁcant. Scale bar 10µm.

Chapter Four

Discussion

JAK2 is widely expressed in multiple tissues, and is activated by multiple cytokine receptors (Behrmann et al., 2004). Upon activation, JAK2 phosphorylates multiple cellular targets, leading to diverse physiological responses to cytokines. JAK2 associates with more than 50 different proteins and specific substrates of JAK2 may be determined by the subcellular localization of JAK2 (Sandberg et al., 2004). JAK2 localizes to the plasma membrane, the cytoplasm, the endoplasmic reticulum, and the nucleus (Behrmann et al., 2004; Lee& Duhe 2006; Lobie et al., 1996; Moulin et al., 2003;

Ram and Waxmann, 1997; Lavoie et al., 2000; Dawson et al., 2011). Additionally, JAK2 associates with both soluble tubulin and insoluble assembled microtubules, and as a result

JAK2 phosphorylates both α- and β-tubulin in response to growth hormone (Ma and

Sayeski, 2007). Previous work revealed JAK2-positive spots, resembling centrosomes, but these structures were not discussed in that report (Ma and Sayeski, 2007). Here we demonstrated that inactive JAK2 localizes to the centrosome, furthermore, our data demonstrated that active JAK2 is a mother centriole component that partially colocalizes with ninein in a cell cycle dependent manner. JAK2 is required for proper microtubule anchoring at the centrosome. In vitro, JAK2 directly phosphorylates the N-terminus of ninein, while the C-terminus of ninein inhibits JAK2 kinase activity, suggesting a co- regulatory association between these proteins. In vivo, overexpressed WT or C-terminal ninein reduced PRL and IFNγ induced JAK2 tyrosyl phosphorylation and STAT1 and

STAT5 tyrosyl phosphorylation. In addition, JAK2 knockdown causes multiple mitotic

defects. Depletion of JAK2 also lead to pre-mature centrosome splitting in interphase cells, suggesting JAK2 may play a role in centrosome cohesion.

The most well-known role the centrosome is involved in the organization and nucleation of cytoplasmic microtubules. The major protein involved in coupling microtubule nucleation and anchoring at the centrosome is ninein, a component of the subdistal appendages located on the mother centriole (Mogensen et al., 2000; Delgehyr et al., 2005). Our studies demonstrate that JAK2 binds and directly phosphorylates ninein, and depletion of JAK2 significantly attenuated microtubule anchoring at the centrosome.

This suggests that JAK2 may regulate ninein-dependent microtubule anchorage mechanisms. Ninein has been proposed to serve as a scaffolding protein for anchoring critical proteins to the centrosome (Chen et al., 2003; Cheng et al., 2007). Even though ninein serves very important functions at the centrosome, little is known about its regulation. Ninein is phosphorylated by GSK3β, Aurora A and PKA on different tyrosines and serine/threonines; however, ninein phosphorylation is tightly regulated

(Howng et al., 2004; Chen et al., 2003; Lin et al., 2006). Centrosomal protein CGI-99 blocks GSK3β phosphorylation of ninein and Aurora A binding protein (AIBp) blocks ninein phosphorylation by both Aurora A and GSK3β (Howng et al., 2004; Chen et al.,

2003; Lieu et al., 2010). Additionally, ninein is tyrosyl phosphorylated in response to vascular endothelial growth factor (VEGF) during tubular morphogenesis of endothelial cells (Matsumoto et al., 2008). Human ninein contains 20 tyrosine residues and only 10 are putative tyrosine phosphorylation sites (Mastumoto et al., 2008). Our studies demonstrate that JAK2 phosphorylates the N-terminus (1-246) of ninein and we are working to determine specific JAK2 phosphorylation sites in ninein. It is intriguing to 62

speculate that the different levels of tyrosyl phosphorylation of the different sites in ninein affect its ability to bind to the SH2 domain of JAK2 and regulate it. Our data supports the idea that ninein not only serves as a scaffolding protein but also regulates

JAK2 activity (Figure 17).

Figure 17. Model of JAK2 and ninein interaction at the centrosome. Ninein localizes to the subdistal appendages of the mother centriole through its C-terminal end and ninein is responsible for anchoring microtubules to the centrosome. In addition, ninein is also thought to act as a scaffolding protein at the centrosome. Our studies demonstrate that JAK2 localizes to the centrosome and phosphorylates the N-terminus of

ninein, while the C-terminal end of ninein is able to inhibit JAK2 kinase activity. This supports the idea that ninein acts a scaffolding protein and can also regulate JAK2 kinase activity (modified from Lin et al., 2006).

We demonstrated that C-terminal ninein inhibits JAK2 kinase activity in vitro and co- expression of C-terminal ninein with activated JAK2 reduced JAK2 tyrosyl phosphorylation in vivo. Additionally, overexpression of WT- and C-terminal ninein decreased JAK2 and subsequently STAT1 and STAT5 PRL- and IFNγ-induced tyrosyl phosphorylation. Silencing of ninein enhanced JAK2 kinase activity. Taken together these data demonstrate that ninein regulates JAK2 activity, impacting downstream signaling pathways. The mechanism by which ninein controls JAK2 kinase activity remains unclear, and therefore further studies will investigate this mechanism. For now, we hypothesize that WT- or C-terminal ninein binding to JAK2 results in a conformational change resulting in JAK2 kinase activity inhibition.

Since our data demonstrated pJAK2 localizing to the mother centriole in the absence of ninein in mitosis, we hypothesize that pJAK2 may participate in ninein- independent functions. We showed that JAK2 participates in centrosomal cohesion, which allows the duplicated centrosomes to function as a single MTOC during interphase. The two duplicated centrosomes are tethered together at their proximal ends by a flexible linker that is composed of C-Nap1, rootletin, Cep68, CEP215-DDK5RAP2 and SIK2 (Mayor et al., 2000; Bahe et al., 2005; Yang and Li, 2006; Graser et al., 2007).

The duplicated centrosomes remain tether together until the onset of mitosis when C-

Nap1 and rootletin are phosphorylated by Nek2 allowing dissociation of the linker components (Fry et al., 1998; Helps et al, 2000). Once untethered, the kinesin-related motor Eg5 separates the centrosomes (Bertran et al, 2011). Further experiments will test possible direct interactions of JAK2 with proteins implicated in centrosomal cohesion and separation to uncover relevant molecular mechanisms. 64

The centrosome is a multifunctional organelle serves many functions including acting as a cell cycle regulator involved in entry to mitosis, anaphase onset, cytokinesis and G1 to S transition, a regulator of cell shape, polarity, motility and organelle transport, and organization of signal transduction pathways (Doxsey et al., 2005; Nigg and Stearns,

2011). Currently, there are nearly 600 centrosomal proteins identified by proteomic studies and in two databases; however this number is continuously growing (Nogales-

Cadenas et al., 2009; Ren et al., 2010; Andersen et al., 2003; Jakobsen et al., 2011).

Many of these centrosomal proteins include kinases, phosphatases, signaling substrates, cyclins, and cell cycle regulators, indicating that the centrosome may serve as a scaffold promoting interactions between different regulatory components. Additionally, a variety of JAK2 substrates and regulators have been identified, including phosphorylated

STAT1, STAT3, and STAT5, SOCS-1, tyrosine kinase Pyk2, Fyn kinase, PI3K, protein phosphatase PTP-BL, and BRCA1 (Takaoka et al., 1999; Ley et al., 1994; Kapeller et al.,

1993; Herrmann et al., 2003; Hsu and White, 1998). These observations support the idea that the centrosome serves as a scaffold for JAK2-dependent signaling transduction pathways (Figure 18). We demonstrated that upon PRL stimulation, activated JAK2 localizes to the centrosome, where it interacts with ninein. Therefore, we hypothesize that through interaction with ninein, activated JAK2 is localized to the centrosome during interphase. However, ninein might have a dual role on the centrosome, functioning as both an anchoring protein and a negative modulator of JAK2 signaling. Therefore, cell surface signals may transit through the centrosome, making it an integrative center of cellular signal transduction pathways. In addition, the centrosome is centrally located in the cell; it is near the nucleus, linked to microtubules, and connected to the Golgi 65

Figure 18. The centrosome as the center of signal transduction The centrosome is centrally located in the cell, and the number of proteins associated with the centrosome is continuously growing. Even though JAK2 is mainly thought to associate with cytokine receptors at the plasma membrane, we have shown that upon cytokine stimulation, JAK2 localizes to the centr osome. In addition, many JAK2 substrates, including STAT1, STAT3, STAT5, PI3K, and known phosphatases such as

PTP-BL and SOCS-1, have been identified at the centrosome. Therefore, it is possible JAK2 signal transduction may occur at the centrosome. For example, active JAK2 localizes to the centrosome and may phosphorylate STATs or PI3K, allowing them to perform their downstream functions. JAK2 activity may then be down-regulated by ninein, PTP-BL, or SOCS. Taken together, cell surface signals may transit through the centrosome, making it an integrative center of cellular signal transduction pathways.

apparatus and proteasome. Location of the centrosome makes it an ideal organelle to serve as a central hub for processing signals from outside the cell these signals into cellular functions. Additionally, multiple fusion proteins have been found at the centrosome in patients with myeloproliferative disorders, including ninein-PDGFRB and

PCM1-JAK2 fusion proteins (Vizmanos et al., 2004; Reiter et al., 2005; Murati et al.,

2005; Bousquet et al., 2005). It was suggested that the oligomerization of the chimeric

PCM1-JAK2 protein at the centrosome may be inducing its constitutive activation.

Interestingly, JAK2 activation in the bone marrow derived pro-B Ba/F3 cell line led to increased centrosome and ploidy abnormalities (Plo et al., 2008). Our data is just beginning to reveal the relationship between the centrosomal protein ninein and JAK2.

In summary, activated JAK2 partially colocalizes with ninein at the mother centriole, and phosphorylates the N-terminus of ninein and in turn the C-terminus of ninein inhibits JAK2 kinase activity (Figure 2). Overexpressed WT- and C-terminal ninein decreased tyrosyl phosphorylation of STAT1 and STAT5 in response to different physiological stimuli. Additionally, JAK2 is required for properly anchored microtubules at the centrosome, a ninein-dependent mechanism. Depletion of JAK2 leads to an increase in mitotic defects and premature centrosome separation in interphase cells. This study has identified both a new site of JAK2 localization and a novel regulator of JAK2 activity, providing insight into the mechanism by which the cell modulates JAK2 activity and thereby its response to cytokine stimulation.

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